In Vitro Methods For Detecting Cytotoxicity: UNIT 2.6

In Vitro Methods for Detecting Cytotoxicity UNIT 2.
The protocols provided in this unit are methods for determining cytotoxicity, a term which
includes the effects toxicants can have on cell growth, metabolic functioning, or viability.
These tests have value because they can provide a general indication of the sensitivity of
cells to toxicants, making them appropriate for screening purposes. Although not specific
indicators of the mechanisms associated with toxicant-induced effects, positive results
with cytotoxicity tests indicate that physiologic or morphologic changes have occurred
in exposed cells. These tests, therefore, may suggest other endpoints to be investigated to
provide more specific mechanistic information about the interactions of toxicants and
cells.
Cytotoxicity is generally used as an inclusive term for changes that can become irre-
versible and that can ultimately affect cell viability. This is true even for cytotoxicity tests
that determine effects on cell growth or metabolic functioning because, with increased
exposure to the toxicant, changes in growth and metabolism, too, can become irreversible
and cell viability can be decreased.
This unit includes protocols for cytotoxicity determinations but does not provide protocols
for culturing cells (see APPENDIX 3B) or protocols for exposure of cells to toxicants. These
procedures vary widely both with cell type and with toxicant. Because cytotoxicity testing
in vitro can be done efficiently and economically, one or more of the methods described
in the sections that follow may be used to screen chemicals for the possibility they might
be toxic before tests are done in animals. This screening could be followed by in vitro
and/or in vivo determination of endpoint alterations more specific to the toxicant in
question and/or to the organ(s) affected. The in vitro cytotoxicity tests themselves alone
cannot be used independently to provide indication of hazard to man or animals.
Seventeen protocols that can be used to indicate cytotoxicity in vitro are included in this
unit. They fall into three groups. First are tests primarily used to indicate the toxicant has
altered cell viability. These tests are valuable for initial general screening. They can also
be used in combination with endpoint assays more specific to the toxicant or target tissue
to provide indication if the more specific change occurs in cells at concentrations below
concentrations needed to decrease viability. This group includes: trypan blue dye uptake
(see Basic Protocol 1); lactate dehydrogenase (LDH) leakage (see Basic Protocol 2 and
Alternate Protocols 1 to 4); neutral red dye retention (see Basic Protocol 3 and Alternate
Protocol 5); and propidium iodide (see Basic Protocol 4) or fluorescent marker (see
Alternate Protocol 6) attachment to double-stranded nucleic acids.
Second, there are tests to indicate the toxicant has effects on cell growth and cell
proliferation. These tests are generally more sensitive to lower concentrations of toxicants
than tests affecting cell viability. These tests can also be used at concentrations of toxicant
that do not necessarily cause irreversible cell damage. Therefore, they could be used to
determine if recovery occurs when the cells are no longer exposed to the toxicant. This
group includes: cell counts (see Basic Protocol 5); [3H]thymidine uptake (see Basic
Protocol 6); and cell cycle analysis with propidium iodide (see Basic Protocol 7).
Finally there are tests to indicate the toxicant has affected cellular metabolic processes.
These tests may be useful to indicate if the toxicant is likely to target the cell membrane,
an intracellular organelle, or a biochemical process. They can be used along with viability
tests to determine concentration and time sensitivities. This group includes MTT dye
conversion by mitochondria (see Basic Protocol 8); alamar blue reduction by respiring
cells (see Basic Protocol 9); and fluorescent dye loss (see Basic Protocol 10) and Assessment of
Cell Toxicity
Contributed by Marion Ehrich and Lioudmilla Sharova 2.6.1
Current Protocols in Toxicology (2000) 2.6.1-2.6.27
Copyright © 2000 by John Wiley & Sons, Inc. Supplement 3
[3H]2-deoxy-D-glucose loss (see Alternate Protocol 7) from cells with compromised
membranes.
The procedures for cytotoxicity included in this unit are a sampling of those available;
they do not provide an all-inclusive list. The protocols do, however, include tests that differ
by indicators of irreversible cell damage and by technical complexity and efficiency.
NOTE: All solutions and equipment coming into contact with cultured cells must be
sterile, and aseptic techniques should be used accordingly.
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2
incubator unless otherwise specified.
BASIC TRYPAN BLUE UPTAKE TO ASSESS VIABILITY

PROTOCOL 1
Trypan blue uptake is commonly determined as cells are counted. This vital dye only
enters dead cells (the cell membrane must be damaged before the dye can enter). Live
cells exclude the dye; therefore, this procedure is used to estimate the percentage of viable
cells in an incubate (also see APPENDIX 3B).
Materials
Cells treated with toxicant, vehicle, or 10% (v/v) Triton X-100 in suspension
Phosphate-buffered saline (PBS; see recipe), pH 7.2 to 7.4
0.1% (w/v) trypan blue (see recipe)
Hemocytometer
Phase-contrast inverted microscope
Hand counter
1. Prepare cells treated with toxicant, vehicle, or 10% (v/v) Triton X-100 in suspension.
Cells that have attached to culture dishes need to be disassociated. This can be done by
removing the medium, adding a solution of trypsin/EDTA (see recipe) for 1 to 5 min,
centrifuging, and removing the trypsin solution (see APPENDIX 3B).
Cells that are treated with 10% Triton-X100 are permeabilized and serve as a positive
control.
2. Wash cells one to three times with PBS, preparing a final resuspension in an exact
volume of PBS (e.g., 1.0 ml, 10.0 ml).
The final resuspension provides the cell concentration counted on the hemocytometer.
3. Place a clean, dry hemocytometer slide on a microscope stage; apply a cover slip over
the chamber so it rests equally across both overflow slots. Set the microscope to use
the 10× objective and focus until the grid lines on the hemocytometer are clear.
The hemocytometer grid has nine large squares, each with an area of 1 mm2. The large
squares are subdivided into smaller squares. (For example, each of the four corner squares
contains sixteen smaller squares; see Fig. A.3B.1.) The depth of the curved chamber
underneath the grid is 0.1 mm, so the volume of cell suspension under each of the nine
large squares is 0.1 mm3 or 10−4 ml.
4. Mix equal volumes of cell suspension and 0.1% trypan blue solution (e.g., 25 µl of
each). Let the mixture stand for no longer than 3 min.
This can be done in the well of the 96-well plate, or on any smooth hydrophobic surface
(e.g., Parafilm, glass slide). Mixing can be done with a micropipet. The cell suspension/dye
mixture should not stand for more than 3 min to prevent dye uptake by viable cells. Trypan
In Vitro Methods blue may be used in concentrations between 0.08% and the 0.4% solution commercially
for Detecting available (Sigma). The cell suspension-to-dye ratio may differ from 1:1 (e.g., 1:2, 1:3, 1:4),
Cytotoxicity but must be appropriately accounted for in the calculation step given below.
2.6.2
Supplement 3 Current Protocols in Toxicology
5. Slowly fill the hemocytometer chamber with the cell suspension/dye mixture by
placing the tip of a micropipettor into the notch under the coverslip. Dispense slowly,
allowing capillary action to draw the cell suspension into the chamber. Do not overfill
or underfill the hemocytometer chamber.
6. Using the 10× objective of the microscope and a hand counter, count the stained
(dead) and unstained (live) cells in one or more of the large squares.
The total area counted needs to have an area of ≥1 mm2; the total number of cells
counted needs to be >100.
7. To calculate the cell concentration in the cell suspension (cells/ml), the total number
of cells counted is divided by the total number of large squares counted and this
number is multiplied by 2 × 104 (to account for the 0.1 mm3, or the 10−4 ml volume
of cells in a large square on the hemocytometer and the 2-fold dilution made when
the trypan blue was added).
8. To calculate the percentage of viable cells, divide the number of viable (unstained)
cells by the total number of cells counted (unstained + stained) and multiply by 100.
LACTATE DEHYDROGENASE (LDH) LEAKAGE TO ASSESS VIABILITY BASIC

PROTOCOL 2
This procedure is used as an indicator of increased membrane permeability and sub-
sequent cell death. The increase in permeability associated with cell death causes release
of the cytoplasmic enzyme, lactate dehydrogenase (LDH), which is present in mammalian
cells. LDH is membrane impermeable and remains within live cells. Kits are available
for LDH detection that decrease the technical complexity of this assay (Sigma).
Materials
Cells
Test compounds: toxicant, vehicle, and Triton X-100
LDH Reagent Kit (DG 1340K, Sigma) containing:
NADH, 0.194 mmol/liter in phosphate buffer
Phosphate buffer, 54 mmol/liter, pH 7.5
Pyruvate, 6.48 mmol/liter in phosphate buffer
96-well microtiter plates, for cell culture
Microtiter plate spectrophotometer with wavelength at 340 nm
1. Set up a 96-well microtiter plate with 1 to 2 × 104 cells/100 µl/well or 100 µl
medium/well (controls). Incubate for 18 to 48 hr to allow cells to attach and reach
60% to 80% confluency. Replace medium for adherent cells; for suspension cells,
proceed directly to step 2.
Use plates coated for cell culture. Use plates with flat-bottomed wells for attached cells
and round-bottomed wells for cells in suspension.
The plate should include wells with medium only to correct for background (e.g., serum
and phenol red in medium), untreated and vehicle-treated cells (negative controls) to
correct for spontaneous release of LDH, and cells treated with 10% Triton X-100 to kill all
the cells (positive control) and allow estimation of the total possible LDH release.
2. Treat cells in remaining wells with test compound, using several concentrations and
several time periods for incubates. Include a vehicle control.
3. To microtiter wells on a second microtiter plate, add 250 µl of NADH solution (0.194
nmol/liter NADH; 54 mmol/liter phosphate buffer, pH 7.5). For suspension cells, add
10 µl of toxicant-treated cells (>104 cells/ml) or control cells to these wells. For Assessment of
Cell Toxicity
2.6.3
Current Protocols in Toxicology Supplement 3
attached cells, add 10 µl of the supernatant of toxicant-treated or control cells to the
wells. Mix.
4. Initiate the reaction by adding and mixing 25 µl of a solution of 6.48 mmol/liter
pyruvate.
5. Record the decrease in absorbance at 340 nm at 3 to 5 min intervals over a period of
time (up to 1 hr).
The change in absorbance needs to be determined over sufficient time to obtain a
meaningful value (change of >0.1 absorbance units over the time observed).
6. After subtracting the LDH release in the negative control from the experimental
incubation and the positive control, compare the rate of decrease (change in absor-
bance/min) to that of the positive control (10% Triton-X-100-treated cells). Express
the results as change in absorbance units/min (∆A340/min).
ALTERNATE IN SITU LDH ASSAY TO ASSESS VIABILITY

PROTOCOL 1
This assay is done by initially putting 10 µl of a 7 mM (0.8 mg/ml) solution of pyruvate
in PBS in the microtiter well with 1 to 2 × 104cells (attached or suspension) in 100 µl
medium and initiating the reaction with 20 µl of a freshly prepared 4.5 mM (3 mg/ml)
NADH solution in PBS. Also, endpoint rather than kinetic assays can be used. To do this,
terminate the reaction by adding 20 µl of the LDH inhibitor oxymate [138 mM (16.6
mg/ml) oxamic acid, sodium salt, in PBS] to microtiter wells containing a 130 µl incubate
of pyruvate, cells, and NADH solution. Read the absorbance at 340 nm.
ALTERNATE PHENYLHYDRAZINE COLORIMETRIC LDH ASSAY TO ASSESS

PROTOCOL 2 VIABILITY
Colorimetric assays can also be used for LDH determinations. One of these procedures
measures the phenylhydrazone produced when pyruvic acid not converted to lactic acid
is quantified as it undergoes reaction with 2,4-dinitrophenylhydrazine. The endpoint is
measured at any wavelength between 400 and 550 nm after 60 min of incubation at 37°C.
This protocol can be performed using Diagnostic Kit 500 (Sigma). The choice of
wavelength depends on the optional wavelength, which is determined prior to the
beginning of the experiment.
ALTERNATE INT COLORIMETRIC LDH ASSAY TO ASSESS VIABILITY

PROTOCOL 3 Another colorimetric LDH assay uses the tetrazolium dye 2-p-iodophenyl-3-nitrophenyl
tetrazolium chloride (INT), with absorbance measured at 490 nm. This can be done as an
endpoint, rather than as a kinetic assay.
Materials
1 to 2 × 105 cells/ml treated with toxicant, vehicle, or 10% (v/v) Triton X-100
36 mg/ml lactate in 10 mM Tris⋅Cl, pH 8.5 (APPENDIX 2B)
INT dye (see recipe)
NAD+ solution (see recipe)
Phosphate-buffered saline (PBS; see recipe), pH 7.2 to 7.4
16 mg/ml oxymate (oxamic acid, sodium salt) in PBS
Microtiter plate reader at 490 nm
1. Set up microtiter plate and treat cells (see Basic Protocol 2, steps 1 and 2).
In Vitro Methods 2. Aliquot 100 µl of cells to each well of a microtite plate.
for Detecting
Cytotoxicity 3. Add 20 µl freshly prepared 26 mg/ml lactate in 10 mM Tris⋅Cl, pH 8.5.
2.6.4
4. Add 20 µl 2 mg/ml INT dye.
5. Add 20 µl NAD+ solution to each well. Incubate 20 min at room temperature.
6. After the incubation, stop the reaction by adding 20 µl of 16.6 mg/ml oxymate at
specific time points <1 hr.
7. Read the absorbance at 490 nm (A490).
8. Calculate the percentage of cells lysed by subtracting the change in absorbance in
negative control cells from the change in absorbance in experimental cells, dividing
by the total absorbance in positive control cells, and multiplying by 100.
KINETIC INT COLORIMETRIC LDH ASSAY TO ASSESS VIABILITY ALTERNATE

PROTOCOL 4
The colorimetric assay described in Alternate Protocol 3 can also be done as a kinetic
assay. For this procedure, mix 100 µl of cells (105/ml) with 100 µl of LDH substrate (see
recipe), adding the substrate mixture to each well at 3-sec intervals. Read absorbance at
490 nm at 3 to 5 min intervals.
NEUTRAL RED DYE RETENTION TO ASSESS VIABILITY BASIC

PROTOCOL 3
Neutral red is a vital dye. It is taken up and retained in viable cells that adhere to the plastic
of microtiter plates. This colorimetric assay can be done in 96-well microtiter plates,
meaning the assay can be conducted simultaneously on a number of incubates containing
cells and various concentrations of toxicant (Babich and Borenfreund, 1992; Veronesi and
Ehrich, 1993).
Materials
105 cells/ml cell suspension
Test compound: toxicant, vehicle, or positive control (1% w/v SDS or 1% w/v
saponin)
Phosphate buffered saline (PBS; see recipe)
1% (w/v) SDS
Neutral red solution (see recipe)
Dye extractor solution (see recipe)
Flat-bottomed, 96-well microtiter plates, cell culture coated
Blotter paper (e.g., absorbent paper towels or filter paper)
Plate mixer
Microtiter plate reader, 550 nm
1. Place 2 × 104 cells (0.2 ml of 105 cells/ml) in wells of a flat-bottom 96-well microtiter
plate. Incubate for a sufficient time to assure attachment and 60% to 80% confluency
(e.g., 18 hr to 3 days).
These plates may need specific coating (e.g., collagen or laminin) for certain cell types that
do not adhere well.
2. Replace medium to include up to 1% (v/v) of toxicant, vehicle, or positive control
solution/well (20 µl per total volume of 200 µl in wells). Expose cells to 1% (w/v)
sodium dodecyl sulfate solution (SDS) or 1% (w/v) saponin solution as a positive
control.
The positive control is included to ensure that the assay is working and to determine the
range of absorbance for the experiment.
Assessment of
Cell Toxicity
2.6.5
3. Incubate cells for time previously designated in the experimental protocol (e.g., 4, 8,
12, 24, 36, 72 hr).
Note that the neutral red assay can be done in the same microtiter plate as the incubation
with toxicant provided the cells are not too confluent and the presence of the test
compound(s) does not significantly decrease cell numbers. If the cells are too confluent to
be a monolayer or if the cells lose adherence, replate to a uniform density before proceeding
to step 5.
4. After incubation with test compounds, remove the medium from the wells by
pipetting. Gently wash the cells adhering to the plate twice with 100 µl PBS. Air dry
the plates to increase adherence of cells to the plate.
5. Then add 40 µl freshly prepared neutral red solution to each well and incubate the
plate for 90 min at 37°C.
6. Remove the neutral red solution and gently but rapidly wash the wells 2 times with
100 µl 37°C PBS. Remove the PBS and blot dry on absorbent paper.
7. Extract the dye from the remaining cells by adding 100 µl dye extractor solution per
well. Mix plate on plate mixer at room temperature for 20 min.
8. Read absorbance on the microtiter plate reader at 550 nm. Compare neutral red
retention in cells exposed to toxicant and those exposed to the vehicle (which is used
to provide the comparative number for 100% viable cells).
A standard curve of neutral red may be prepared to provide results in microgram per
milliliter. Plates can be reread the next day, if needed, as the neutral red in the cells is
stable overnight.
ALTERNATE NEUTRAL RED ENDPOINT ASSAY TO ASSESS VIABILITY

PROTOCOL 5
This assay can be done with endpoints either in the visible range (550 nm) or as a
fluorescent assay (488 nm excitation, 645 emission). First the cells are treated with
toxicant, vehicle, or positive control.
For cells in suspension in test tubes rather than microtiter wells, incubate 5 × 106/ml cells
with 0.036% (w/v) neutral red (prepared in commercially available Hank’s balanced salt
solution) at 37°C for 30 min in a humidified 37°C, 5% CO2 incubator. Then centrifuge
cells 10 min at 400 × g, 4°C, and wash the cell pellet twice with medium centrifuging 10
min at 400 × g between washings. After washing resuspend and dilute cells in medium to
5 × 106 cells/ml. Mix 100 µl cells with 100 µl of 0.1 M acetic acid containing 1% (w/v)
SDS, dilute to 1.25 ml with normal saline and read the absorbance at 540 nm or
fluorescence at 645 nm with 488 nm excitation.
Alternatively, for cells in suspension, incubate 100 µl cells (1 to 2 × 105 cells/ml) with
0.1% (w/v) neutral red for 3 hr at 37°C in microtiter plates, then wash with PBS by adding
200 µl PBS to each well, mixing by pipetting, and centrifuging the plate for 10 min at
1000 rpm in a centrifuge with a plate carrier. Carefully remove the PBS. Repeat the PBS
wash two to three times. Next add 1% glacial acetic acid/50% methanol to extract the dye
and fix the cells. Read absorbance at 540 to 550 nm or fluorescence at 645 nm with 488
nm excitation.
In Vitro Methods
for Detecting
Cytotoxicity
2.6.6
PROPIDIUM IODIDE ATTACHMENT TO DOUBLE-STRANDED NUCLEIC BASIC
ACIDS TO ASSESS VIABILITY PROTOCOL 4
The fluorescent dye propidium iodide (excitation 530 nm, emission 645 nm) is excluded
from living cells, but enters and combines with double-stranded nucleic acids (i.e., DNA)
of dead cells. The assay for cell viability using propidium iodide attachment to double-
stranded nucleic acids can be done simultaneously with determination of membrane
integrity using a variety of membrane-permeant fluorescent probes (e.g., CFDA-AM,
which has an excitation 485 nm and emission 530 nm) because the fluorescent spectra do
not overlap (Nieminen et al., 1992).
Materials
0.5 × 105 cells/ml
Test compounds: toxicant, vehicle, and positive control
Phosphate buffered saline (PBS; see recipe)
376 µM digitonin solution
30 µM propidium iodide dye (Molecular Probes)
Flat bottomed, 96-well microtiter plates, for cell culture
Fluorescence microtiter plate reader
1. Plate cells (0.5 × 104/well) and allow to attach overnight before exposing to toxicant.
Incubate with toxicant at predetermined concentrations and times. Include wells for
positive and negative controls on each microtiter plate.
2. Wash plate free of medium. Add 10 µl of 376 µM digitonin solution to positive control
wells. Add 100 µl propidium iodide (30 µM) to all wells.
3. Incubate plate for 30 min at 37°C.
4. Read in a microtiter plate fluorescence spectrophotometer set with excitation at 530
nm and emission at 645 nm.
Alternatively, excite at 488 nm.
5. Compare wells exposed to test compounds and wells exposed only to vehicle
(negative control) to wells exposed to digitonin (100% cell death; the positive
control).
The positive control will provide wells with the highest fluorescence.
6. Calculate per cent of viable cells as fluorescence in test wells less negative control
divided by fluorescence in digitonin-treated wells less negative control, with the
quotient multiplied by 100.
USING FLUORESCENT MARKERS TO ASSESS VIABILITY ALTERNATE

PROTOCOL 6
Several kits that use binding of fluorescent markers to nucleic acids as an indicator of loss
of cell viability are available from Molecular Probes (Haugland, 1996). For example, the
Live/Dead Assay includes calcein-AM, a membrane-permeable dye, which is cleaved by
intact cells to calcein (fluoresces green) and ethidium homodimer, a membrane-imper-
meable dye that labels nucleic acids of membrane-compromised (nonviable) cells to
produce a red fluorescence. With excitation of 488 nm, emission at 530 nm will indicate
live cells and emission at 585 nm will indicate dead cells.
Assessment of
Cell Toxicity
2.6.7
BASIC ASSAYS TO ASSESS CELL GROWTH
PROTOCOL 5
Cell counting is done along with determination of viability using trypan blue and is
described under Basic Protocol 1. Another indicator of cytotoxicity is the loss of ability
of certain cell types to extend processes (extensions >2× the length of the cell body) when
exposed to agents that promote process development (e.g., retinoic acid; Abdulla and
Campbell, 1993). A change in expression of specific proteins can also be an indication of
cytotoxicity and forms the basis for kits manufactured by Xenometrics.
Estimation of cells counts can also be made using protein determinations, such as the
Kenacid Blue dye binding method recommended by the European Fund for Replacement
of Animals in Medical Experiments (FRAME; O’Hare and Atterwill, 1995).
Materials
Cells (1 to 2 × 105 cells/well) or 150 µl of 1 × 106 cells/ml, treated with toxicant,
vehicle, or control in microtiter plates
Phosphate-buffered saline (PBS; see recipe), 37°C
Fixative: 1% (v/v) glacial acetic acid/50% (v/v) ethanol/49% (v/v) water
Kenacid blue dye (see recipe)
Washing solution: 10% (v/v) ethanol/5% (v/v) glacial acetic acid/85% (v/v) water
Microtiter plate reader, 577 nm
1. After cells have been exposed to toxicant, vehicle, or control solution, remove
medium and rinse cells one to three times with warm PBS.
2. Add 150 µl fixative solution to each well and fix 20 min.
3. Remove fixative solution and add 150 µl freshly prepared Kenacid working solution.
Shake the plate 2 to 3 hr.
4. Wash wells twice with 150 µl wash solution.
5. After last wash, fill the wells with wash solution and shake for 20 min until the dye
has completely gone into solution.
6. Read absorbance at 577 nm using a well with no cells as a reference well.
The absorbance of the reference well which contains no cells should be zero when the
spectrophotometer is set at 404 nm. Absorbance of wells with cells could be read at 600
nm rather than at 577 nm.
BASIC [3H]THYMIDINE UPTAKE TO ASSESS CELL GROWTH

PROTOCOL 6
This procedure can be used as an indication of cytotoxicity because toxicants can decrease
the rate of cell division. The assay can also be used as an indicator of cell proliferation,
as [3H]thymidine incorporates into the nucleic acids of proliferating cells. This is a
relatively complex, yet sensitive procedure that requires used of radiolabeled chemicals.
Materials
1 to 15 × 105 cells/ml
Serum-free chemically-defined medium (e.g., RPMI 1640 or DMEM), 37°C and
ice-cold
Test compounds(s): toxicant and vehicle
20× [3H]thymidine solution (see recipe)
10% (w/v) trichloroacetic acid (TCA) solution, ice-cold
In Vitro Methods
0.3 N sodium hydroxide
for Detecting Scintillation fluid
Cytotoxicity continued
2.6.8
24-well plates, coated for cell culture
Liquid scintillation counter
Radioactive waste disposal system
CAUTION: When working with radioactivity, take appropriate precautions to avoid
contamination of the experimenter and surroundings. Carry out the experiment and
dispose of wastes in an appropriately designated area following the guidelines provided
by your local radiation safety offices (also see APPENDIX 1A).
CAUTION: Trichloroacetic acid is very caustic. Handle it with care.
NOTE: All incubations should be performed at 37°C using an incubator or water bath.
1. Place 1 ml cells in 24-well tissue culture plates and incubate 6 to 24 hr to allow cells
to attach.
Cells with density of 1 to 15 × 105/ml may be used.
2. Wash medium from wells and replace with 1 ml serum-free, chemically defined
medium 24 to 48 hr before treating cells with test compounds.
Alternatively, cells (e.g., lymphocytes) can be collected from toxicant-treated animals.
3. For in vitro exposure to toxicants, add toxicant or vehicle, 50 µl/well, to cells for the
times specified in the testing protocol.
4. Add 50 µl/well sterile 20× [3H]thymidine solution for the last 2 hr of incubation with
the test substance. Continue to incubate at 37°C.
For certain cell types (e.g., lymphocytes), incubation with [3H]thymidine may be as long
as 72 hr. When incubation is done for very short periods of time (e.g., 15 min), the
concentration of [3H]thymidine incubated with cells should be increased (e.g., 2.5 ìCi/ml).
Other components of nucleic acids may also be used, such as BrdU (bromo-deoxyuridine)
in a relatively similar procedure. [3H]leucine and [3H]glycine incorporation, which are
used as indicators of the rate of protein synthesis, would also be expected to provide similar
results.
5. Stop the reaction by placing plates on ice. Wash cells 3 times with 1 ml of ice-cold
serum-free medium, then carefully remove all medium by vacuum aspiration.
Remember that the medium and washes are radioactive and dispose of appropriately.
6. Add 1 ml ice-cold 10% trichloroacetic acid (TCA) to each well. Leave plates on ice
for 10 min to allow precipitation of DNA and protein. Remove trichloroacetic acid
by aspiration.
This aspirate may be discarded or counted for determination of unincorporated
[3H]thymidine.
Rather than using the caustic TCA solution, cells may be harvested by centrifugation or
harvested by using a cell harvester with fiberglass filters (the filters are subsequently placed
in vials with scintillation fluid and counted).
If cells are collected by centrifugation, wash once with warm conditioned medium (medium
in which cells have been grown), and recentrifuge at low speed (e.g., centrifuge a 1- to 2-ml
sample 3 min at 430 × g at 37°C in pre-warmed 12-ml glass conical centrifuge tubes).
Following centrifugation, remove the supernatant, and air dry the pellet before resuspend-
ing for scintillation counting. Alternatively, the pellet may be resuspended in 0.5 ml
hypertonic solution (e.g., 1.8% saline), placed on a slide for histological examination, fixed
by sequential exposure for 10 min each to 5% to 7% trichloroacetic acid (at 5°C), 70%
ethanol at room temperature, and 95% ethanol at room temperature, and air-dried. For
example, the Kodak Emulsion slidemailer system could be used. After the cells are fixed
and air-dried, the slides are covered with emulsion, exposed, developed according to the Assessment of
manufacturer’s instructions and analyzed using a microscope. Cell Toxicity
2.6.9
7. Add an additional 1 ml of 10% TCA to the plates and leave on ice for an additional
5 min. Wash a third time with 10% TCA and leave on ice an additional 5 min to
complete the precipitation. Remove the TCA.
8. Solubilize the precipitated protein with 0.5 ml of 0.3 N sodium hydroxide by
incubating for 30 to 60 min at room temperature. Transfer 250 µl to a miniscintillation
vial with 5 ml scintillation fluid and count.
Concentration and length of incubation with sodium hydroxide may be increased (e.g., to
1 N and to 24 hr) to increase digestion of the cells.
Although [3H]thymidine uptake is usually the endpoint, [3H]thymidine release could also
be measured. With this system, incubate cells with [3H]thymidine (concentration and time
of incubation predetermined by protocol), wash three times, then resuspend at 4 × 104
cells/well in 24-well tissue culture plates in 1.0 ml medium. Following overnight incubation
at 37°C in 5% CO2, aspirate the medium, mix aliquots with scintillation fluid, and count.
Cytotoxicity (in per cent) is calculated by comparing the radioactivity counted in the
sample and control wells.
BASIC CELL CYCLE ANALYSIS WITH PROPIDIUM IODIDE TO ASSESS CELL

PROTOCOL 7 GROWTH
This procedure can be used as an indicator of cytotoxicity for toxicants that affect DNA
synthesis and, therefore, cause cell arrest mostly in the G1/G0 phase of the cell cycle.
Some of these cells may undergo apoptosis later, whereas others may have decreased rates
of cell growth and proliferation.
Propidium iodide (PI), a DNA-binding fluorochrome, is the most commonly used DNA
dye for cell cycle analysis. Because it can be excited at 488 nm, it can be used on most
common flow cytometers. Its stoichiometric binding to double-stranded nucleic acids
allows fluorescent intensity (>620 nm) to be used as an indicator of cellular DNA content.
Using propidium iodide is now a common procedure for DNA analysis (Krishan, 1975).
Materials
Toxicant-treated and control cells in suspension
Standard azide buffer (see recipe)
Vindelov’s PI (see recipe)
12 × 75–mm clear polystyrene tubes
30-µm mesh (optional)
Flow cytometer
1. Harvest cells, centrifuge 5 min at 1000 × g, decant supernatant, and vortex pellet.
2. Resuspend cells in 1 ml standard azide buffer, count the cells, and recentrifuge 5 min
at 1000 × g.
3. Resuspend cells in standard azide buffer so that cell concentration is 106/ml.
4. Transfer 1 ml of this suspension to another tube and add 0.5 ml of Vindelov’s PI.
5. Let cells sit at 4°C for 5 min to 1 hr, then filter through 30-µm mesh if necessary to
remove noncellular particulate material.
Filtering is optional.
6. Analyze cells by flow cytometry using 488 nm excitation, gating out doublets and
In Vitro Methods clumps using pulse processing, and measuring fluorescent emission at >620 nm.
for Detecting
Cytotoxicity
2.6.10
For cell proliferation determinations, the CyQUANT Cell Proliferation Assay Kit is
available from Molecular Probes. Fluorescence increases when the dye binds to nucleic
acids. To perform this procedure, plate cells at 103 to 104 cells per well in 200 ìl medium
in 96-well plates. Incubate the cells 6 to 18 hr to allow the cells to attach, replace the
medium, and treat the cells with toxicant. Then remove culture medium from cells, freeze
and then thaw the cells, add the reagent and cell lysis buffer provided in the kit, then
measure fluorescence with 485 ± 10 nm for excitation, and 530 ± 12.5 nm emission. The
manufacturer states that this method is sensitive enough to detect fluorescence in cells at
a concentration as low as 50 cells/ml.
MTT DYE CONVERSION TO ASSESS METABOLIC CAPABILITY BASIC

PROTOCOL 8
This assay requires cells that are actively able to metabolize 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) to an insoluble formazan precipitate. The
assay is used as an indicator of mitochondrial function in live cells; however, loss of
activity cannot be used to distinguish compromised cells from dead cells. As an indicator
of cytotoxicity, MTT results have been reported to be comparable in sensitivity to those
obtained using [3H]thymidine incorporation (Mosman, 1983). The assay can be used with
cultured cells or with tissues, as the colored product is solubilized and extracted from the
test system. Because the dye needs to be extracted from the cells, this procedure cannot
be used to provide cell counts.
Materials
5 × 104 cells/ml cell suspension
Test compound: toxicant or vehicle
Culture medium
5 mg/ml MTT in PBS (store 2 to 3 weeks at 4°C or longer at −20°C)
Dimethyl sulfoxide (DMSO), photometric grade
Sorensen’s buffer (see recipe)
96-well microtiter plates, for cell culture
Aspiration system for removal of medium from cells
Micropipettor (20 to 200 µl) with sterile micropipettor tips
Microtiter plate reader
1. Plate cells at 1 × 104 cells/well by adding 200 µl of a 5 × 104 cells/ml suspension to
each well of a 96-well tissue culture plate. Include control wells with medium and
no cells. Incubate overnight at 37°C 5% CO2.
Test compounds (toxicant and vehicle) may be added before or after this incubation.
Use plates with flat-bottomed wells for adherent cells and round-bottomed wells for
suspension cells.
2. Carefully aspirate off the medium, avoiding removal of cells. If not previously
incubated with toxicant, replace medium with fresh medium (200 µl) containing up
to 0.1 ml of test substance. Include wells with medium without cells or test compound,
medium without cells but with test compound, cells without test compound (but with
test compound vehicle) as controls. Incubate at 37°C, 5% CO2, for predetermined
times (e.g., several hours, overnight, and/or for several days).
3. After incubation with test compound, carefully aspirate off medium and replace with
medium and 20 to 50 µl MTT solution for a total volume of 200 µl. Incubate for 4 to
6 hr at 37°C, 5% CO2.
4. Carefully remove the MTT-containing medium. Add 200 µl DMSO with 25 µl
Sorensen’s buffer per well. Mix the plate until the formazan crystals are dissolved. Assessment of
Cell Toxicity
2.6.11
Rather than DMSO, 200 ìl of 0.04 N HCl in isopropanol may be used to solubilize the
formazan product made from the MTT dye.
A 10% (w/v) solution of SDS in 0.01 M HCl rather than DMSO can be used to lyse the cells
during an overnight incubation at 37°C.
5. Read plate on microtiter plate reader at 550 to 570 nm.
If medium contains phenol red, readings may need to be compared to those taken at 660
nm, which is a wavelength where quenching by the medium is minimized.
6. Compare absorbance in wells containing the test substance with wells containing
untreated control cells.
Provided appropriate concentrations are used, a concentration-response curve can be
constructed.
BASIC ALAMAR BLUE REDUCTION TO ASSESS METABOLIC CAPABILITY

PROTOCOL 9
This bioassay uses a water-soluble indicator of cytotoxicity, eliminating the need for the
washing and fixation steps needed in other assays using vital dyes (e.g., neutral red, MTT).
As dye extraction is not done, the time needed for data collection is decreased. Data may
be collected as either fluorescence (excitation at 530 to 560 nm; emission at 590 nm) or
absorbance at 570 or 600 nm. Either cells adhering to the plastic of microtiter plates or
cells in suspension culture can be used for this assay. Since the dye is nontoxic to the cells,
the same cultures can be monitored over a series of time points. This assay can be used
for cell proliferation determinations as well as for cytotoxicity assays. The dye and
reagents for this assay are available in a kit from Biosource International, whose technical
literature states that it provides results that compare favorably with the MTT assay.
Materials
Cells in microtiter plates
Alamar blue assay kit (Biosource International)
Microtiter plate reader
Additional reagents and equipment for counting cells and assessing viability using
trypan blue dye (Basic Protocol 1)
1. Do preliminary experiments to establish optimal cell density and incubation time for
the cells used for testing.
2. Harvest cells in log-phase growth; count and estimate viability with trypan blue (see
Basic Protocol 1).
3. Plate cells using the number that provides a predetermined optimal density (e.g., 200
µl of 1 × 104 cells/ml for some cancer cell lines) and at cell numbers that provide
optical densities above and below optimal. Allow cells to grow at 37°C, 5% CO2, for
sufficient time that they reach log phase (e.g., 2 to 3 days).
4. Aspirate growth medium from the wells and add 200 µl of new medium containing
up to 10% volume of test compound to the wells.
These cells should be incubated for selected periods of time with several concentrations of
test compound and the plate should include some wells in which the medium contains only
vehicle.
5. Aseptically add an amount of alamar blue equal to 10% of the culture volume (e.g.,
In Vitro Methods
for Detecting 20 µl in a well that will contain 200 µl total or 22 µl to a well containing 200 µl
Cytotoxicity medium).
2.6.12
6. Return cultures to the incubator for selected incubation times (e.g., from several hours
to several days).
For all data points data can be collected immediately after the incubation or plates can be
stored overnight at 4°C and read the next day. Cell proliferation can be measured once or
several times over a 3- to 5-day time period. The assay should be stopped (by refrigeration
at 4°C) when cells reach 80% to 90% confluence.
7. Measure fluorescence (excitation 530 to 560 nm; emission 590 nm) or absorbance
(at 570 and 600 nm) in a microtiter plate reader at desired time points.
If using absorbance, subtract the background at 600 nm from the absorbance measured at
570 nm.
Appropriate time points should be determined by preliminary experiments analyzing the
growth of the cells.
8. Calculate cytotoxicity as absorbance (or fluorescence) in treated cells over absor-
bance (or fluorescence) in control cells. Generate a graph that plots log concentration
of test compound on the x axis and percent decrease in absorbance (or fluorescence)
from control on the y axis. Compare dye reduction over time in wells containing test
compound and control wells.
MEASURING FLUORESCENT DYE LOSS TO ASSESS METABOLIC BASIC

CAPABILITY PROTOCOL 10
The acetoxymethyl ester derivative of 5-carboxyfluorescein diacetate (CFDA-AM) is one
of several membrane-permeant dyes available from Molecular Probes that can be used to
indicate membrane integrity (Haugland, 1996). For CFDA-AM and other such dyes,
membrane integrity is indicated because the product formed upon esterase-induced
cleavage will not leave cells if the cell membrane is intact. The ability to leak CFDA is
considered an early indicator of cell damage. This assay for cytotoxicity can be performed
at the same time as the propidium iodide assay for cell viability (see Basic Protocol 4),
as the spectra for fluorescence do not overlap (Nieminen et al., 1992).
Materials
Cells
50 µg/ml CFDA-AM in PBS (prepared fresh)
Phosphate-buffered saline (PBS; see recipe)
Fluorescence microtiter plate reader
1. Add 0.5 × 104 cells to each well of a 96-well plate. Allow to attach overnight. Incubate
with test compounds according to the experimental protocol.
2. Remove medium, wash cells with PBS, and add 100 µl of a 50 µg/ml solution of
CFDA-AM.
Other membrane-permeable dyes may be used, such as the calcein-AM that is included in
the Live/Dead Viability/Cytoxicity Assay of Molecular Probes.
3. Incubate cells for 30 min in a 37°C incubator.
4. Read fluorescence using excitation at 485 nm and emission at 530 nm.
The microtiter plate fluorescence reader should be set to a sensitivity that provides 1000
to 2000 fluorescence units per 1 to 2 × 104 cells/well.
5. Compare fluorescence in wells treated with toxicant with that of wells containing
cells but no test compound or vehicle.
Assessment of
Cell Toxicity
2.6.13
ALTERNATE USING [3H]2-DEOXY-D-GLUCOSE TO ASSESS METABOLIC CAPABILITY
PROTOCOL 7
Another means of determining cell membrane integrity is by exposing cells to [3H]2-de-
oxy-D-glucose, which is membrane permeable. This substrate will be converted to
membrane impermeant [3H]2-deoxy-D-glucose-6-phosphate within living cells, and will
remain within the cells if the membrane is intact (O’Hare and Atterwill, 1995).
Materials
106 cells grown in 60-mm culture dishes
Phosphate-buffered saline (PBS; see recipe)
0.55 µCi/ml [3H]2-deoxy-D-glucose in PBS
1 mg/ml glucose in PBS
1 M NaOH
1 M HCl
Scintillation fluid
CAUTION: When working with radioactivity, take appropriate precautions to avoid
contamination of the experimenter and surroundings. Carry out the experiment and
dispose of wastes in an appropriately designated area following the guidelines provided
by your local radiation safety offices (also see APPENDIX 1A).
1. Grow 106 cells in 60-mm culture dishes to the appropriate state of confluency.
The appropriate state of confluency corresponds to a number estimated for each type of
cell, which is determined before starting the experiment.
2. Remove the growth medium, and wash the cells twice with 5 ml PBS each wash.
3. Add 5 ml of 0.55 µCi/ml [3H]2-deoxy-D-glucose in PBS. Incubate for 2 hr to load
cells.
4. Remove incubation solution and wash 3 times with 5 ml PBS per wash.
5. Add 2 ml PBS containing 1 mg/ml glucose and the appropriate concentration of test
compound. Incubate for predetermined times (e.g., 6, 12, and 24 hr).
6. At up to three time points during the incubation, take 50-µl samples and add the
samples to 3 ml scintillation fluid.
7. At the end of the incubation, remove the medium from the cells. Add 1 ml 1 M NaOH
and incubate for 30 min to dissolve the cells.
8. Add the cell lysate to 3 ml scintillation fluid.
9. Rinse the dish with 1 ml 1 M HCl and add the rinse to 3 ml scintillation fluid.
10. Count the samples, lysate, and rinse. Calculate concentration depletion of radioac-
tivity over the time of incubation.
The values obtained will provide a means to differentiate what remains within and what is
released by the cells.
This procedure may also be done using a patented polycarbonate perfusion block (O’Hare
and Atterwill, 1995).
In Vitro Methods
for Detecting
Cytotoxicity
2.6.14
REAGENTS AND SOLUTIONS
Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Dye extractor solution

Citrate buffer: Mix 21.01 g/liter citric acid with 200 ml 1 M NaOH. Bring volume
to 1 liter with water and check that pH is 4.2. To 60 ml of this solution add 40 ml of
0.1 M HCl. Store 2 to 3 months at 4°C.
Working solution: Mix citrate buffer 1:1 (v/v) with absolute alcohol.
INT dye
Stock solution: Dissolve tetrazolium dye INT (2-p-iodophenyl-3-nitrophenyl tetra-
zolium chloride) at a final concentration of 40 mM (20 mg/ml) in DMSO to provide
a stock solution that can be stored in a tightly closed bottle for 2 to 3 months at room
temperature.
Working solution: Dilute the stock 1:10 (v/v) with PBS (see recipe) to prepare a 4
mM (2 mg/ml) working solution just before doing the assay.
Kenacid blue dye
0.2 g Coomassie brilliant blue R-120
125 ml ethanol
315 ml H2O
Store for 1 to 2 months at 4°C
Kenacid Blue working solution: Add 3 ml glacial acetic acid to 22 ml Kenacid blue
dye solution immediately before use.
LDH substrate
5.2 × 10−2 M L(+) lactate
6.6 × 10−4 M INT (2-p-iodophenyl-3-nitrophenyl tetrazolium chloride)
2.8 × 10−4 M phenazine methosulfate
1.3 × 10−3 M NAD+
0.2 M Tris⋅Cl, pH 8.2 (APPENDIX 2A)
Prepare just before assay
NAD+ solution
4.5 mM NAD+ (3 mg/ml)
13.5 U/ml diaphorase
0.03% (w/v) bovine serum albumin
1.2% sucrose in PBS, pH 8.2
Prepare just before assay
Neutral red stock solution
Prepare 0.1% (w/v) neutral red in distilled water, autoclave, and acidify with 2 drops
of glacial acetic acid per 100 ml. Store up to 3 months in a dark container at 4°C.
Working solution: Dilute stock solution 1:10 (v/v) with PBS (see recipe) or 1:10
with 1.8% NaCl immediately before use.
Phosphate buffered saline (PBS)
For 1 liter:
8 g NaCl (0.138 M final)
0.2 g KCl (3 mM final)
1.15 g disodium monophosphate (Na2HPO4; 8 mM final)
0.2 g potassium dihydrophosphate (KH2PO4; 2 mM final
H2O to 1 liter
Assessment of
continued Cell Toxicity
2.6.15
Adjust pH to 7.2
Filter sterilize through a 0.4-µm filter
Store up to 3 months at 4°C
Sorenson’s buffer
0.1 M glycine
0.1 M NaCl
Adjust pH to 10.5 with NaOH
Store up to 2 months at 4°C, but check pH before use
Standard azide buffer
10 gram FA Bacto buffer (Difco 2314-15-0)
10 ml of 10% (w/v) sodium azide solution
10 ml heat-inactivated fetal bovine serum (FBS)
980 ml distilled water
Adjust pH to 7.2 ± 0.05
Store in foil-wrapped bottle up to 1 month at 4°C
[3H]thymidine solution, 20×
20 µl of 1 mCi/ml [3H]thymidine
980 µl sterile, serum-free medium
Prepare fresh on the day of assay
For the assay 50 ìl of the stock is diluted to 1000 ìl with serum-free medium to give
1 ìCi/ml in well.
Trypan blue, 0.1% (w/v)
Dilute 1 ml commercially available 0.4% (w/v) trypan blue solution (Sigma) with
3 ml PBS (see recipe) or balanced salt solution (Sigma). Store tightly closed up to
6 months at room temperature.
Trypan blue in concentrations of 0.08% to 0.4% (w/v) can be used to determine percentage
of viable and dead cells seen on the hemocytometer. With 0.1% trypan blue the cells are not
as darkly stained and are easier to visualize.
Trypsin/EDTA
To prepare 100 ml of trypsin 0.05% (w/v) with 0.02% (w/v) EDTA, add 2 ml of 10×
(2.5%) trypsin (Sigma) and 0.133 ml of 15% (0.5 M) EDTA, pH 8.0, to 97.87 ml of
PBS (see recipe). Keep refrigerated, but warm to 37°C before use.
A ready-made solution is available from Sigma and Life Technologies (Gibco). The trypsin
solution can be kept up to 4 weeks at 4°C. If it is not used frequently, it can be stored > 1 yr
frozen at −5° to −20°C.
Vindelov’s propidium iodine stain (PI)
1.21 g Tris base (0.01 M final)
584 mg NaCl (10 mM final)
10 mg ribonuclease (700 U final)
50.1 mg PI (7.5 × 10−5 M final)
1.0 ml Igepal CA-630 (Sigma; 0.1% final)
1 liter H2O
Mix until dissolved
Adjust pH to 8.0
Filter through a 0.45-µm filter
Store in foil-wrapped bottle up to 1 month at 4°C
CAUTION: Propidium iodide is toxic and potentially carcinogenic; handle with care and
In Vitro Methods label solution appropriately.
for Detecting
Cytotoxicity
2.6.16
COMMENTARY
Background Information having effects on more specific endpoints at
All of the protocols provided in this unit can concentrations lower than those that cause gen-
be used to detect damage to cells following in eral cell damage. Time-response studies are
vitro application of toxicants to cells. In addi- also useful in cytotoxicity determinations be-
tion, several of the procedures can also be used cause they, too, indicate relative sensitivities
as in vitro determinants of cytotoxicity for between irreversible cell damage and func-
cells derived from animals tested with toxi- tional changes.
cants. The endpoints are nonspecific. In other The investigator should recognize that
words, they can occur in many types of cells higher concentrations or longer times of expo-
damaged under a wide variety of situations, sure may be needed for expression of some
including exposure to chemicals, anoxia, and endpoints than others (e.g., loss of cell viability
physical agents. Cytotoxicity tests, therefore, versus alteration of cell growth), that some
cannot be expected to be used to define specific toxicants may require metabolic activation be-
mechanisms of injury induced by specific toxi- fore they are cytotoxic, and that it is difficult to
cants. Some tests, however, can suggest a sub- compromise metabolic function without caus-
cellular structure (e.g., the nucleus, mitochon- ing subsequent loss of viability. In fact, any of
dria, membranes) affected by particular toxi- the protocols provided could be used as indica-
cants, and more specific tests could then be tors of irreversible cell damage. Some of the
performed to better describe the toxicant ef- protocols, however, especially those that indi-
fects (Barile, 1994; Forsby et al., 1995). Al- cate cell growth, can be used at concentrations
though the protocols provided in this unit have of toxicants that are low enough for recovery
been grouped according to one of three pri- to occur if the toxicant is removed.
mary endpoints (viability, cell growth, meta- Cytotoxicity assays can be used with cells
bolic capability), all can be used to indicate of various tissue origin; however, the sensitivity
dose-related toxicant-induced changes that to toxicant-induced cytotoxicity may vary with
can eventually result in cell death. For investi- the cell type used for testing. Such differences
gators hoping to use such tests to indicate could provide the basis for studies that deter-
toxicant-induced effects without having toxi- mine tissue-specific sensitivities to toxicants.
cant-induced cell death, a prudent first choice For example, if cells that originate in the nerv-
among the protocols for cytotoxicity would be ous system are more sensitive than cells of other
among those without viability as a primary tissue origin (e.g., hepatic, kidney), it may be
endpoint, with inclusion of concentration and possible to suggest that the toxicant in question
time-response effects in the investigation. As could target the nervous system. However, cer-
an example, the investigator interested in func- tain cell types used for cytotoxicity determina-
tional disruption rather than cell death could tions (e.g., those of neoplastic origin, regardless
examine concentration-related changes in me- of tissue origin) may be very hardy, and con-
tabolic capability as a more reasonable end- centrations required for cytotoxic effects may
point than viability (e.g., for neurotoxicants or may not be physiologically relevant.
and reversible hepatotoxicants). The investigator may choose an assay
Concentration response is an important part method based on endpoint (e.g., viability, me-
of cytotoxicity determinations and should be tabolic effects), sensitivity, ease of manipula-
included to provide indication of the range over tion for multiple incubates, time needed, and
which toxicant-induced cytotoxicity or re- availability of equipment. Indications are that
sponse occurs. This is especially important if all procedures presented in detail in this unit, if
comparisons are to be made between irre- used with sufficiently high concentrations of
versible effects indicative of cytotoxicity and toxicants, may be reasonably expected to detect
any other, more specific, functional, biochemi- comparable cytotoxicity (irreversible cell dam-
cal, or morphological changes that occur after age), without indication of toxicant-specific
exposure to the toxicant. If concentrations for mechanistic effects on cell function. Therefore,
cytotoxicity and other alterations are similar, choice of protocol would be determined by the
the toxicant-induced general cytotoxicity may other factors listed above, rather than by effec-
contribute to the changes in all endpoints de- tiveness at detecting cytotoxicity alone. For
termined. If a functional change occurs at lower example, if large numbers of tests are to be
concentrations than cytotoxicity, the change done, the colorimetric assays with the vital dyes
may be reversible, and the toxicant may be neutral red, MTT, and alamar blue may be the Assessment of
Cell Toxicity
2.6.17
test systems of choice because these procedures comparisons of dose- and time-related changes
can all be done in 96-well microtiter plates. If in cell growth and proliferation assays for cy-
the investigator wishes to observe toxicant ef- totoxicity may be made with dose- and time-
fects on the same cells over a period of time, related changes in viability.
the alamar blue procedure may be chosen. This
is because, among the vital dyes, only alamar Assays for cell viability
blue forms a water-soluble product; with neu- The trypan blue procedure (Basic Protocol
tral red and MTT the assay needs to be termi- 1) is commonly used because it not only pro-
nated to extract the colored product formed by vides indication of how many cells are alive,
cellular metabolic processes. Some assays can but also how many live cells are being used for
be used at lower and higher toxicant concentra- any subsequent experiment. This quantitative
tions, respectively, to determine cell prolifera- test is very simple and results can be rapidly
tion as well as cytotoxicity (e.g., alamar blue, obtained. The technique requires no specific
MTT, propidium iodide, [3H]thymidine incor- training or specialized equipment.
poration), with the latter radiometric assay, al- Measurement of LDH activity (Basic Proto-
though more complex, more sensitive than sim- col 2 and Alternate Protocols 1 to 4) is another
ple addition of alamar blue for this particular indicator of cell viability. The enzyme activity
endpoint. The fastest and simplest method for is measured external to the cells, as it leaks from
cytotoxicity, trypan blue uptake, is too labori- dead cells. LDH activity can be measured in the
ous for precision if large numbers of samples ultraviolet range (340 nm), based on the loss of
are to be examined for cytotoxicity. Some cy- NADH due to its oxidation to NAD+ as pyru-
totoxicity assays are available in kit form, de- vate is converted to lactate. If a colorimetric
creasing their technical complexity (e.g., LDH; assay is preferred, a colored phenylhydrazone
fluorescent assays that measure attachment of produced when 2,4-dinitrophenylhydrazine is
markers to nucleic acids in dead cells or indicate incubated with the remaining pyruvate can be
membrane permeability; alamar blue; stress measured.
gene assays). The availability of kits as well as LDH release can also be measured as NAD+
the detailed technical information included is reduced when lactate is converted to pyru-
with such kits may make them the protocols of vate. The resultant production of NADH then
choice in certain situations. reduces FAD coupled to diaphorase with sub-
For investigators initiating studies on the sequent transfer of FADH2 electrons to the
cytotoxic effects of toxicants, testing is best tetrazolium dye INT (2-p-iodophenyl-3-ni-
done in the cells of tissue origin relevant to the trophenyl tetrazolium chloride).
investigation. In other words, cytotoxicity test- Neutral red (Basic Protocol 3 and Alternate
ing for neurotoxicants should be done in cells Protocol 5) is a third assay for cell viability.
of nervous system origin; cytotoxicity testing Neutral red is a weakly cationic dye that is
for hepatotoxicants is best done in cells of bound to anionic sites on intracytoplasmic
hepatic origin. Comparisons, however, should vacuoles and granules. It accumulates in
be made between sensitivities in cells of various lysosomes and other intracellular vacuoles of
origin if the toxicant is to be identified by its living cells. Ability to retain this dye is lost
effects on a specific organ or system (e.g., a when cell die. Neutral red needs to be extracted
neurotoxicant or a hepatotoxicant). Dose- and from the cells; to do so requires that cells be
time-response investigations should be in- lysed. This procedure, therefore, cannot be used
cluded. Dose- and time-related endpoint deter- like trypan blue exclusion to quantitate the
minations specific for the tissue in question or number of live cells taking up the dye.
for the toxicant target, if known, are useful to Propidium iodide retention (Basic Protocol
determine if changes occur at concentrations 4) is another method for determining loss of
lower than those that cause general cytotoxic- cell viability. This fluorescent dye passes
ity. Examples of specific endpoints are the toxi- through compromised cellular membranes and
cant-induced inhibition of enzymes responsi- then intercalates between base pairs in double-
ble for neurotransmitter degradation in neuro- stranded nucleic acids. This agent has different
nal cells, toxicant-induced changes in the signals when free or bound in cells, with emis-
quantity of a protein marker for astrocytes, loss sion at >600 nm detecting the bound species
of intercellular adhesion in cells with barrier that is formed once it passes through the com-
function, and alteration of extracellular mark- promised membranes of dead cells. Experi-
In Vitro Methods
for Detecting ers in lymphocytes. In the absence of an end- ments by Nieminen et al. (1992) indicated that
Cytotoxicity point that indicates a specific cellular function, results of assays using propidium iodide for
2.6.18
determining cell viability correlated with the assay is best performed on cells that have active
results obtained by measuring lactate dehydro- mitochondria. It, therefore, can be used as an
genase (LDH) release. indication of cellular proliferation, and it is far
easier to perform than [3H]thymidine incorpo-
Assays for cell growth ration.
The number of cells in a growing culture Estimation of cytotoxicity using the alamar
(Basic Protocol 5) can be decreased by expo- blue technique (Basic Protocol 9) involves in-
sure to toxicants. This may or may not be due corporation of an oxidation-reduction (redox)
to loss of viability of a certain portion of the indicator that both fluoresces and changes color
cells. Cell numbers can be indicated directly, in response to the reduction that occurs in
by counting the cells, or indirectly, by measur- medium as the cells grow. Reduction occurs in
ing protein levels, and comparing results to the presence of FMNH2, FADH2, NADH,
cultures that have not been treated with the NADHPH, and cytochromes. The reduction of
toxicant. the dye requires that it be taken up by the cells
Proliferating cells incorporate [3H]thymid- and participate in cellular respiratory proc-
ine (Basic Protocol 6) into nucleic acids, com- esses. The water-soluble dye is nontoxic, and
peting with unlabeled thymidine. If the toxicant growing cells, which provide a reducing envi-
inhibits cell proliferation, with or without af- ronment, cause the indicator to change from a
fecting cell viability, the incorporated radioac- non-fluorescent to a fluorescent product.
tivity will be decreased. Compromised membranes affect metabolic
Like cell counting or [3H]thymidine incor- functioning of cells and may be used to indicate
poration, cell cycle analysis (Basic Protocol 7) cytotoxic effects of toxicants. Membrane dam-
is another means for determining the effects of age may lead to cell death. Fluorescent dye loss
toxicants on cell growth and differentiation. (Basic Protocol 10) indicates that membrane
Alterations may or may not be due to effects on leakage is occurring. These assays make use of
cell viability. Cell cycle analysis using the fact that cells in culture contain nonspecific
propidium iodide is based on the fact that DNA esterases. The basic protocol provided is based
content is not the same during different phases on the principle that CFDA-AM, which is able
of the cell cycle. It is normally diploid in G1. to cross cell membranes, will be hydrolyzed by
When cells start to synthesize new DNA in the intracellular esterases to CFDA, which does not
S phase, DNA content will be greater than cross the membrane. The CFDA will remain
diploid. After DNA synthesis is completed and within the cells and fluoresce, unless mem-
the cell enters the G2 phase, it has a tetraploid brane integrity is impaired and it leaks out.
DNA content. During mitotic division (M Similarly, loss of [3H]2-deoxy-D-glucose from
phase), the DNA content comes back to normal cells (Alternate Protocol 7) is also an indication
(diploid). If the DNA is stained with a DNA- of loss of membrane integrity.
specific dye, such as propidium iodide, and the
DNA content per cell is estimated by flow Critical Parameters and
cytometry, the G1/S/G2 ratio can be evaluated Troubleshooting
and compared between untreated and toxicant- These protocols alone may not provide all
treated cells. the information needed to obtain optimal re-
sults when in vitro methods for cytotoxicity are
Assays for metabolic capability used. Additional details are, therefore, provided
Still other protocols have been included be- for each of the protocols in the paragraphs that
cause they are reputed to indicate cytotoxicity follow.
because they measure change in the metabolic When using typan dye exclusion (Basic Pro-
functioning of the cell. Cell functions can be tocol 1) to estimate viable cell concentrations,
compromised by toxicants, and this compro- it is important that the cell suspension be ho-
mise could result in loss of viability. The MTT mogenous or erroneous results will be obtained
colorimetric assay (Basic Protocol 8) deter- as the relatively small volume counted is mul-
mines the ability of viable cells to convert the tiplied by the total volume of cells available.
soluble tetrasodium salt MTT [3-(4,5-di- Even in the small volume of cells used for this
methylthiazol-2-yl)-2,5-diphenyl tetrazolium procedure, a relatively large number of cells
bromide] to an insoluble formazan precipitate. must be counted (>100), and some means for
The salts initially formed can be reduced within recording differential counts is needed, such as
the cell to blue-colored formazan crystals that using a double-scale hand counter for live and Assessment of
can be visualized on a spectrophotometer. The dead cells or a hand counter for live cells and Cell Toxicity
2.6.19
pencil marks for dead cells. A decision needs be a reasonable alternative. Another alternative
to be made before starting as to how to count would be to grow the cells in a different type
cells that fall on lines (i.e., count cells touching of serum and/or different medium.
the lines at the top and left of any square but Care must be taken with cell cultures, be-
omit cells touching the lines on the bottom and cause high spontaneous release of LDH can
right sides of the square). occur in the case of suboptimal cell culture
Cell suspensions on which trypan blue ex- conditions (e.g., when cell density is too high
clusion is to be measured should be placed on or when cell damage occurs during transfer of
the hemocytometer so they are not too concen- cells or addition of medium).
trated or counting will be difficult. If it appears Low overall absorbance values may occur
that >10% of the cells are clustered, the suspen- during LDH determinations, especially during
sion should be redispersed by vigorous pipet- colorimetric assays, if substrates are degraded
ting and the suspension recounted. by light.
For trypan blue exclusion determinations, it Neutral red uptake (Basic Protocol 3 and
is important not to let the cell suspension stand Alternate Protocol 5) is a technically simple
with the dye too long, as viable cells may then method for determination of cell viability but
begin to take up the trypan blue. Also, trypan use of this assay still requires caution. For
blue has a greater affinity for serum protein than example, neutral red solution can form crystals,
for cellular protein; therefore, counting cells especially when prepared in phosphate buffers;
suspended in PBS is preferred to counting cells such crystals are unlikely to form if the solution
in serum-containing media. is prepared in distilled water whether the solu-
The trypan blue uptake and cell counting tion is stored at 4°C or at room temperature.
procedure can be tedious if a large number of If the neutral red assay is done in wells in
cell suspensions need to be counted. Further- which cells were incubated with toxicant, care
more, the hemocytometer needs to be clean and must be taken when removing the medium from
dry before counting is done. A hemocytometer cells in the microtiter wells before addition of
that is not dry will dilute the sample and under- the neutral red solution. If adhering cells die,
estimate cell counts. Washing with 70% ethanol they may become less attached than control
can decrease the time needed for cleaning and cells. Color would then be less intensive and
drying; remaining ethanol could, however, lead more related to cell number than to cytotoxicity,
to underestimation of viability. which would be underestimated.
When LDH leakage (Basic Protocol 2) is Plates used for the neutral red assay may be
used to indicate loss of cell viability, a number processed twelve wells at a time to ensure
of cautions need to be observed in order to uniformity of adhesion.
obtain good, reproducible results. For kinetic If the cell number in incubates with toxicant
and endpoint assays, the negative control is too high or if the toxicant decreases cell
should produce <5% of the change in absor- numbers, the cells should be replated before the
bance of the positive control. In addition, the neutral red assay is run.
positive control needs to provide a sufficiently Propidium iodide binding (Basic Protocol
high change in absorbance that meaningful 4) to the DNA of dead cells is another indicator
results can be obtained. Furthermore, the reac- of cytotoxicity. For proper use of this protocol,
tion should be conducted under constant tem- optimal conditions for a particular fluorescence
perature conditions to decrease interassay vari- scanner should be determined by preliminary
ability. Since results are based on differences experiments in which both propidium iodide
between control and experimental incubates, concentration and cell number per well are
cell concentrations need to be similar. Prelimi- varied. Concentrations of propidium iodide
nary experiments with untreated cells are useful >50 µM in microtiter wells are unlikely to
for optimization of conditions for a particular further increase fluorescence.
cell type that is to be used for subsequent The propidium iodide assay is more repro-
experiments. ducible with cells that strongly adhere to plastic
Among the problems which could be en- than with cells that stay in suspension.
countered with the LDH assay is the possibility Propidium iodide is a light-sensitive, hygro-
of a high background reading, which may be scopic powder, red to dark red in appearance.
noted when LDH is present in serum-contain- If properly stored (dry at 0° to 4°C), it will
ing medium. If this occurs, decreasing the se- remain stable for ≥4 years. Propidium iodide
In Vitro Methods
for Detecting rum content of the cell culture medium to 5% dissolves readily in water at 1 mg/ml; a higher
Cytotoxicity (provided it does not affect cell viability) may concentration (10 mg/ml) requires heating and
2.6.20
may contain some insolubles. Either water or estimation of DNA content and placement of
PBS can be used for solubilizing propidium cells in the cell cycle. Therefore, ribonuclease
iodide. Solutions are stable for up to 2 years if treatment is critical for the staining. (The
stored at 0° to 4°C in the dark with 0.1% (w/v) RNAase is a part of Vindelov’s solution; how-
sodium azide as preservative. Propidium iodide ever, further incubation of cells with 1 mg/ml
is toxic and possibly carcinogenic, so protective ribonuclease for 5 min at room temperature
clothing should be worn when handling this may be useful in some situations. Use DNAase-
chemical. free ribonuclease, or DNA damage will occur
When cell counts (Basic Protocol 5) are used and that will interfere with the assay.)
to indicate cytotoxicity, cultures that are to be The use of standard azide buffer is recom-
used as control and cultures that are to be mended for assays of phenotypic markers
exposed to the toxicant need to be identical where there is a possibility of capping surface
before the experiment begins. When cell num- receptors when doing cell cycle studies. Azide
ber or quantity of cell protein is used as an prevents capping and allows an assay to be
endpoint, the culture needs to be one that con- performed at room temperature (or higher, if
tinuously divides and multiplies over time. In- necessary). Azide is also a microbial inhibitor.
hibition of growth should follow a concentra- Obtaining quality histograms from the flow
tion response. cytometer during cell cycle analysis may be
Because changes in cell counts or cell pro- difficult. Each cell type may need a slightly
tein may take several days, the toxicant needs different treatment, but saturating concentra-
to be water soluble, relatively stable, and non- tions of propidium iodide always need to be
volatile in order to remain in contact with the used to be sure that all cells are stained.
cells. Toxicants that need metabolic activation The MTT assay (Basic Protocol 8), although
may not cause a response. relatively simple from a technical aspect, may
Cell proliferation using [3H]thymidine (Ba- differ for different cells, as each has different
sic Protocol 6) requires use of radiolabeled metabolic capability. Therefore, preliminary
compounds and use of such compounds re- experiments are needed to establish the optimal
quires special precautions. cell number for each well, the duration of the
Careful pipetting of the [3H]thymidine so- experiment, the time of MTT incubation nec-
lution is needed so wells do not become con- essary in order to provide a final absorbance
taminated. Variability increases if pipetting is that can easily be measured, and conditions to
not precise and if contamination occurs. Large provide control values sufficiently high that
volumes of diluted [3H]thymidine should not loss of mitochondrial function in toxicant-
be prepared; prepare what is needed for the treated cells can be measured. (Number of cells
experiment only. seeded per well, for example, should be suffi-
Medium removed from the plates after the cient to maximize the number of cell doublings
incubation with [3H]thymidine is radioactive. that occur over a toxicant treatment period of 2
A closed collection system that is properly to 5 days.) Once established, these parameters
labeled reduces risk. need to be kept constant over the experiments
[3H]thymidine assays that use trichlo- in which data are to be collected and compared.
roacetic acid (TCA) need to be done with ex- The metabolic state of the cells will affect
treme care because TCA is very caustic. the outcome of MTT assays; the cells should
For cell cycle analysis (Basic Protocol 6), be kept as uniform as possible. Variability
cell membranes need to be permeable to allow among wells on a single plate can occur.
the propidium iodide to enter. This can be done If volumes of medium differ among the
by treating the cells with azide buffer or by wells (e.g., by >10%) during the MTT assay,
using ethanol. The propidium iodide needs to absorbances will also differ. Evaporation needs
enter and stain the DNA in all cells. Other to be controlled to avoid this potential cause of
information about propidium iodide is in the variability. Old medium can decrease the effi-
paragraphs describing Basic Protocol 4. ciency of the assay, causing underestimation of
The propidium iodide used for cell cycle the metabolic capability of control cells. Ab-
analysis binds to double-stranded nucleic acids sorbance can also be affected by the concentra-
through intercalation between base pairs, with tion of D-glucose in the culture medium. It can
no preference for purine or pyrimidine base be expected to be unaffected by pH or phenol
pairs. It does not bind to single-stranded nucleic red concentrations <10 mg/ml.
acids. Propidium iodide also stains double- Some test substances may increase mito- Assessment of
stranded RNA and, thus, can give a wrong chondrial activity; this could be a cytotoxic Cell Toxicity
2.6.21
effect, but would not be indicated by the lower scope. Live cells exclude trypan blue; dead cells
absorbances usually expected when MTT is do not. Control cells should be >85% viable
used to indicate cytotoxicity. (>85 of 100 cells counted not taking up the dye).
MTT does not deteriorate if kept in a light- Provided cell suspensions are homogenous and
proof container at 4°C for several weeks. the procedure is carried out appropriately, re-
The formazan product formed from MTT petitive counting of the same suspension should
needs to be completely solubilized before read- provide counts that do not vary by more than
ing absorbance. 20%.
As with the MTT assay, optimal conditions LDH leakage (Basic Protocol 2 and Alter-
for the alamar blue assay (Basic Protocol 9) nate Protocols 1 to 4) is detected spectro-
may vary with cell line, cell density, and incu- photometrically. Untreated cells should retain
bation time. Preliminary experiments are nec- LDH and have minimal loss over the time the
essary to establish a cell density at a particular assay is run; the greatest loss should be with the
incubation time that provides a linear response positive control, the Triton-X-100-treated cells.
of alamar blue reduction correlating with the An example of results from a study that used
cell density. Preliminary experiments should LDH leakage is provided in Figure 2.6.1 (Song
also be done to determine the incubation time et al., 1997). In this study, LDH leakage was
that provides maximal conversion of dye added used as an indicator of the cytotoxic effects of
to control cells from the oxidized (blue) form MPTP (1-methyl-4-phenyl-1,2,4,6-tetrahy-
to the fully reduced (red) form of the dye. dropyridine), an agent that induces a Parkin-
Although concentration-response curves may sonian-like syndrome in primates, and its me-
be obtained as early as 1 hr after alamar blue is tabolite MPP+. With cultured cells, reproduci-
added, longer incubations are preferred to in- b ility amo ng po sitive con trols and
crease absorbance or fluorescent signals. reproducibility within negative controls should
High cell numbers or extended incubation be within 20%.
times (>24 hr) can cause loss of linearity in The neutral red assay (Basic Protocol 3 and
production of the reduced (red form) of the Alternate Protocol 5) is another spectro-
alamar blue dye; absorbance may not only photometric assay for viability. When this pro-
loose its linearity, but may actually decline tocol is used, absorbance is higher when there
under these conditions. Microbes that may con- are more live cells in a well, so values in control
taminate cultures will also reduce alamar blue. wells of dividing cells depend on time of incu-
Bovine serum concentrations up to 10% bation. The product extracted from the cells is
(w/v) do not interfere with the alamar blue a reddish color. As an approximate estimate,
assay, nor does the presence of phenol red in absorbances of 0.2 to 0.4 could be expected in
the growth medium. Phenol red, however, may wells containing 105 cells/ml. This would be
increase background absorbance by as much as ∼3 µg/ml neutral red. The limit of sensitivity is
0.3 optical density units. Alamar blue itself is estimated to be ∼1000 cells/well; changes in
not cytotoxic at concentrations as high as 25% dye retention with 2000 cells in a well have
with exposures for up to 20 hr. been reproducibly detected (± 20%).
Fluorescent dye loss (Basic Protocol 10) can With binding of propidium iodide to DNA
also be used as an indicator of cytotoxicity. As (Basic Protocol 4), fluorescence is used as an
the membrane-permeable fluorescent dye is an indicator of cytotoxicity. In this protocol for
ester, it needs to be kept dry to avoid spontane- cytotoxicity, untreated cells should show little
ous hydrolysis. fluorescence, as the dye is excluded from nor-
[3H]-2-deoxy-D-glucose retention (Alter- mal cells. The positive control cells, treated
nate Protocol 7) has been used as a means for with digitonin, should show the maximal fluo-
determining cell integrity. However, because rescence. Propidium iodide absorbs blue-green
the compound is radioactive, special precau- light and fluoresces red. Reproducibility
tions are required; thus many laboratories now should be within ± 20%.
prefer to use the fluorescent dyes. Cytotoxicity can also be indicated by fewer
cell numbers (Basic Protocol 5) or less protein
Anticipated Results in cultures exposed to the toxicant. The de-
In vitro methods for cytotoxicity use a vari- crease seen on exposure to the toxicant should
ety of endpoints. For example, assessment of follow a concentration response, with at least
viability using trypan blue exclusion (Basic three points on a concentration-response curve
In Vitro Methods
for Detecting Protocol 1) is done by direct observation. Cell that are significantly different from the cell
Cytotoxicity are counted by viewing them through a micro- number or protein in the control incubate.
2.6.22
Another means for detecting cytotoxicity by counting, discrimination among incubates can
reduction in cell growth or proliferations is by be made even if [3H]thymidine incorporation
the uptake of [3H]thymidine (Basic Protocol 6). is low.
Radioactivity is dependent on the number of Flow cytometry is used when cell cycle
cells in an incubate (Fig. 2.6.2; Ahmed et al., analysis (Basic Protocol 7) provides indication
1994). If cytotoxicity occurs, [3H]thymidine of cytotoxicity. Histograms obtained by flow
uptake should be less in treated cells than in cytometry allow estimation of the percentage
control cells. This assay is quite sensitive, and, of cells in different phases of the cell cycle. If
by increasing the time of liquid scintillation the toxicant interferes with DNA synthesis, the
A
50
* MPTP (M)
45 * 0
10 – 7
40 10 – 6
35 * 10 – 5
% leakage of LDH
10 – 4
30 10 – 3
3 × 10 – 3
25
20
15
*
10 *
** *
5
0
B 50
* MPP* (M)
45 0
* 10 – 7
40
10 – 6
% leakage of LDH
35 * 10 – 5
10 – 4
30 * 10 – 3
*
25 *
*
20
*
15
*
10
*
5 *
0
4.0 8.0 24.0 48.0 72.0 120.0
Exposure time (hr)
Figure 2.6.1 Effects of (A) MPTP and (B) MPP on the LDH leakage in SH-SY5Y human
neuroblastoma cells. After treatment (10−7 to 3 × 10−3 M) for 4, 8, 24, 48, 72, 120 hr. Percent LDH
leakage was determined by comparing to cells treated with Triton X-100 (100%). At the assay
conditions, the LDH activity that leaked from Triton X-100-treated cells was 198.69 ± 12.81
mOK/min; the leakage of LDH from untreated cells was 4.36 ± 0.51% of the Triton X-100 treated
cells. Results were averages of at least 3 individual experiments (mean ± SE). The results for
MPP-treated cells are significantly different from the control (p < 0.05). Reprinted, with permission,
from Song et al. (1997). Intox Press. (See discussion of reference for an explanation of the decrease
in release of LDH after 48 hr.) Abbreviations: MPP, 1-methyl-4-phenyl-pyridine; MPTP, 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine. Assessment of
Cell Toxicity
2.6.23
A 1.4
180,000
1.2
150,000
Specific absorbance ± SEM

1.0
120,000
cpm ± SEM
0.8
90,000
0.6
60,000
0.4
0.2 30,000
0.0 0
B 1.4 180,000
1.2
150,000
Specific absorbance ± SEM
1.0
120,000
cpm ± SEM
0.8
90,000
0.6
60,000
0.4
30,000
0.2
0.0 0
500,000 50,000 5,000 500 50 0.0
Cells/well
Figure 2.6.2 The spontaneous proliferation of YCD3-1 cells at indicated cell numbers per well
was determined by Alamar blue (solid line) and [3H]thymidine (interrupted line) assays. Alamar Blue
and [3H]thymidine were added 3 hr after the culture set up and proliferation determined at 24 (A)
and 48 hr (B) post culture. The specific absorbance was determined by subtracting A600 from A570.
[3H]thymidine incorporation data are presented as mean cpm. Reprinted, with permission, from
Ahmed et al. (1994). Elsevier Science B.V.
In Vitro Methods
for Detecting
Cytotoxicity
2.6.24
Table 2.6.1 Effect of Various Neurotoxicants on Membrane Integrity and Viability of SH-SY5Y
Human Neuroblastoma Cellsa
Test chemical and Percent of control (±SEM) atc

Endpoint
concentration (M)b 4 hr 24 hr
Membrane integrity (CFDA retention) MPTP 10−6 113 (5) 100 (6)
10−5 100 (4) 88 (4)
10−4 93 (4) 89 (1)
10−3 80 (4)d 59 (6)d
MPP+ 10−6 94 (1) 81 (6)
10−5 87 (8) 77 (1)d
10−4 91 (4) 69 (8)d
10−3 71 (9)d 58 (10)d
Viability MPTP 10−6 98 (1) 97 (1)
10−5 98 (1) 97 (1)
10−4 97 (1) 97 (1)
10−3 97 (2) 97 (1)
MPP+ 10−6 98 (1) 97 (1)
10−5 97 (1) 97 (1)
10−4 98 (1) 97 (1)
10−3 98 (1) 98 (1)
aData from Rowles et al., 1995.
bAbbreviations: MPP, 1-methyl-4-phenylpyridine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
cResults presented as percentage of control activity, mean (SEM) of 3 to 5 replicate assays (with wells done in triplicate in each
assay).
dDifferences from control (p < 0.05). Values in untreated cells for retention of CFDA (indicator of membrane integrity) measured
as fluorescence units = 1221 ± 115 for 1 to 2 × 10−4 cells/well (sensitivity 2 on Cytofluor fluorescence plate reader). Viability
(measured with propidium iodide, verified with trypan blue) over all assays = 97% ± 1. Viability determined by binding of
propidium iodide to nuclei of nonviable cells based on comparison with positive control (digitonin). Reprinted, with permission,
from Rowles et al., 1995; Mary Ann Liebert, Inc., Publishers.
majority of the cells will be in the G1 phase, functioning and stop growing. This will lessen
and a very low percentage will be in the S and the reduced environment usually present when
G2 phases. cells are growing, and decrease the amount of
Metabolic capability of cells may be com- fluorescent product produced. Results are com-
promised when they are exposed to toxic sub- parable with other assays using vital dyes (e.g.,
stances. One method for detecting the effects MTT), and reproducibility should be ± 20%.
of toxicants on the metabolism of cells uses Decreased membrane integrity is indicated
MTT, which cells with intact mitochondria con- by fluorescent dye loss (Basic Protocol 10 ) in
vert to a formazan dye which can be detected the wells of the microtiter plate that contain test
spectrophotometrically. The MTT assay (Basic compound and cells. Membrane changes can
Protocol 8) is rapid, versatile, quantitative, and occur at time points earlier or at concentrations
highly reproducible, with a low intratest vari- lower than needed for loss of cell viability, as
ation (± 15% standard deviation). Results are can seen in a study with MPTP and MPP+
expressed as a percentage of control absor- (Table 2.6.1; Rowles et al., 1995). For a fluo-
bance, and cell numbers per well are not quan- rescent dye assay, reproducibility should be ±
tified. Although an indicator of a cytotoxic 20%. An assay which indicates viability (e.g.,
effect on mitochondrial function, the test can- propidium iodide binding, Basic Protocol 4)
not distinguish between cells with reduced ef- may be done at the same time as assays which
fects and those that are dead. indicate membrane compromise (Table 2.6.1;
The alamar blue reaction (Basic Protocol 9) Rowles et al., 1995).
is a spectrophotometric or fluorescent assay
that depends on the number of cells in the Time Considerations
incubate (Fig. 2.6.2; Ahmed et al., 1994). Cy- The trypan blue assay (Basic Protocol 1) is
totoxicity causes cells to decrease metabolic probably the easiest of all protocols included Assessment of
Cell Toxicity
2.6.25
in this chapter. A cell count and viability calcu- increase the time needed to do this assay. Incu-
lation can be done in <5 min per sample. bations of cell suspensions with propidium io-
More time is needed when LDH leakage dide stain (at 4°C) may be overnight or even
(Basic Protocol 2 and Alternate Protocols 1 to longer. To determine cytotoxicity, incubations
4) is used to indicate loss of cell viability. For with the toxicant may require hours to days
this procedure, a microtiter plate which contains before the staining and flow cytometry are
96 wells would provide triplicate wells for over done.
20 samples and the positive and negative con- After incubation with toxicant, 4 to 6 hr are
trols. Pipetting would take ~10 min; the plate required for MTT incorporation (Basic Proto-
may be stabilized by delaying the first reading col 8). An additional hour may be required for
of a kinetic assay for up to 7 min after initiation MTT removal, dye extraction, crystal solubili-
of the reaction. The reaction may run as long as zation, and reading on the microtiter plate.
an hour. With a microtiter plate reader, all wells After incubation with toxicant, the time of
can be read simultaneously; some microtiter the alamar blue assay (Basic Protocol 9) de-
plate readers can be programmed to do the pends on the optimal time for incubation of dye
appropriate calculations. Total time per plate with cells (determined by preliminary experi-
would be expected to be ~2 hr. ments). This can range from 1 to 24 hr.
Cell in microtiter plates previously incu- Using loss of fluorescent dye (Basic Proto-
bated with toxicants could be processed for col 10) to determine toxicant-induced compro-
neutral red retention (Basic Protocol 3 and mise of membrane integrity is a relatively
Alternate Protocol 5) in ∼2.5 hr. The longest straight-forward, simple procedure. After incu-
times are those needed for incubating with the bation of cells and toxicant ∼1 hr should be
dye (90 min) and the time needed for dye sufficient to do this assay.
extraction (20 min). Over 20 samples, and posi-
tive and negative controls, could be run in Literature Cited
triplicate on a single plate. The process can be Abdulla, E.M. and Campbell, I.C. 1993. Use of
interrupted at the air-drying step, with plates neurite outgrowth as an in vitro method of as-
sessing neurotoxicity. In Markers of Neuronal
stored for several days before addition of the
Injury and Degeneration (J.N. Johansson, ed.)
neutral red solution. pp. 276-279. Ann. N.Y. Acad. Sci., 679.
Propidium iodide binding to DNA of dead
Ahmed, A., Gogal, R.M., and Walsh, Y.E. 1994. A
cells (Basic Protocol 4) can also be used to new rapid and simple non-radioactive assay to
indicate toxicant effects on cell viability. For monitor and determine the proliferation of lym-
each plate processed after incubation of cells phocytes: An alternative to [3H]thymidine incor-
and toxicant, ∼1 hr is needed. poration assay. J. Immunol. Methods 170:211-
224.
When counting cells (Basic Protocol 5) to
compare cell growth between control and toxi- Babich, H. and Borenfreund, E. 1992. Neutral red
assay for toxicology in vitro. In In Vitro Methods
cant-treated cells, the time at which the count-
of Toxicology (R.R. Watson, ed.) pp. 237-251.
ing is done is dependent on the growth rate of CRC Press, Boca Raton, Fla.
untreated cells. Doubling time may vary from
Barile, F.A. 1994. Introduction to In Vitro Cytotoxi-
several hours to several days. The procedure cology Mechanisms and Methods. CRC Press,
itself takes little time (see Basic Protocol 1), but Boca Raton, Fla.
the time needed to determine if cytotoxic ef- Forsby, A., Pilli, F., Bianchi, V., and Walum, E. 1995.
fects are occurring may take days. Determination of critical cellular neurotoxic
After incubation for predesignated times concentrations in human neuroblastoma (SH-
with toxicant, [3H]thymidine incorporation SY5Y) cell cultures. Alternatives to Lab. Ani-
mals 23:800-811.
(Basic Protocol 6) may take from 0.25 to 24 hr.
Preparation of cells for scintillation counting Haugland, R.P. 1996. Handbook of Fluorescent
Probes and Research Chemicals, 6th ed. Molecu-
may take 1 to 3 hr, and scintillation counting
lar Probes, Eugene, OR.
will take additional hours.
Krishan, A. 1975. Rapid flow cytometric analysis of
Flow cytometric analysis for cell cycle de-
mammalian cell cycle by propidium iodine stain-
terminations (Basic Protocol 7) may be rela- ing. J. Cell Biol. 66:188-193.
tively rapid (<1 hr), but time is needed for
Mosman, T. 1983. Rapid colorimetric assay for cel-
preparation and interpretation of the graphs lular growth and survival: Application to prolif-
provided. The staining procedure needed be- eration and cytotoxicity assays. J. Immunol.
fore the flow cytometry can be expected to take Methods 65:55-63.
In Vitro Methods
for Detecting ∼30 min. Longer incubations of cells with Nieminen, A.L., Gores, G.J., Bond, J.M., Imberti,
Cytotoxicity propidium iodide can improve results but will R., Herman, B., and LeMaster, J.J. 1992. A novel
2.6.26
cytotoxicity screening assay using a multi-well Borenfreund, E., and Puerner, J.A. 1985. A simple
fluorescence scanner. Toxicol. Appl. Pharmacol. quantitative procedure using monolayer cultures
115:147-155. for cytotoxicity assays (HTD/NR-90). J. Tissue
O’Hare, S. and Atterwill, C.K. 1995. Methods in Cult. Methods 9:7-9.
Molecular Biology 43: In Vitro Toxicity Testing This is an early paper from a laboratory with recog-
Procedures. Human Press, Totowa, N.J. nized expertise with the neural red assay (Basic
Rowles, T.R., Song, X., and Ehrich, M. 1995. Iden- Protocol 3 and Alternate Protocol 5).
tification of endpoints affected by exposure of Nieminen et al., 1992. See above.
human neuroblastoma cells to neurotoxicants at
concentrations below those that affect cell viabil- Optimization of the procedure for measuring cell
ity. In Vitro Toxicol. 8:3-13. viability by propidium iodide uptake (Basic Protocol
4) is described in this reference.
Song, X., Perkins, S., Jortner, B.S., and Ehrich, M.
1997. Cytotoxic effects of MPTP on SH-SY5Y Technical Information, Alamar Blue Assay,
human neuroblastoma cells. Neurotoxicology Biosource International, Camarillo, CA.
18:341-354.
Details about the alamar blue assay system (Basic
Veronesi, B. and Ehrich, M. 1993. Differential cy- Protocol 9) and how to use it are provided.
totoxic sensitivity in mouse and human cell lines
exposed to organophosphate insecticides. Toxi- Technical Information included with LDH Assay
col. Appl. Pharmacol. 120:240-246. Kits, Sigma Chemical, St. Louis, MO.
These provide specific details for use of their kits
Key References when LDH leakage (Basic Protocol 2 and Alternate
Alley, M.C., Scudiero, D.A., Monks, A., Hursey, Protocols 1 to 4) is used as the indicator of loss of
M.L., Czerwinski, M.J., Fine, D.L., Abbot, B.J., cell viability.
Mayo, J.G., Shoemaker, R.H., and Boyd, M.R.
1988. Feasibility of drug screening with panels Technical Information on Trypan Blue, Sigma
of human tumor cell lines using a microculture Chemical, St. Louis, MO.
tetrazolium assay. Cancer Res. 48:589-601. This provides a detailed protocol, including a dia-
This describes a microassay for MTT (Basic Proto- gram of a hemocytometer, for the trypan blue assay
col 8). (Basic Protocol 1).
Application notes for the Cytofluor Fluorescent

Plate Reader, Millipore Corporation, Marlbor-
ough, MA. Contributed Marion Ehrich and
Lioudmilla Sharova
These notes discuss use of multiple probes for mul-
tiple endpoints in a single incubate of cells, provid-
Virginia-Maryland Regional College of
ing excitation and emission data, that can be used Veterinary Medicine
when fluorescent dye loss (Basic Protocol 10) is Blacksburg, Virginia
used to indicate cytotoxicity.
Assessment of
Cell Toxicity
2.6.27

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In Vitro Methods for Detecting Cytotoxicity UNIT 2.

BASIC TRYPAN BLUE UPTAKE TO ASSESS VIABILITY

LACTATE DEHYDROGENASE (LDH) LEAKAGE TO ASSESS VIABILITY BASIC

ALTERNATE IN SITU LDH ASSAY TO ASSESS VIABILITY

ALTERNATE PHENYLHYDRAZINE COLORIMETRIC LDH ASSAY TO ASSESS

ALTERNATE INT COLORIMETRIC LDH ASSAY TO ASSESS VIABILITY

KINETIC INT COLORIMETRIC LDH ASSAY TO ASSESS VIABILITY ALTERNATE

NEUTRAL RED DYE RETENTION TO ASSESS VIABILITY BASIC

ALTERNATE NEUTRAL RED ENDPOINT ASSAY TO ASSESS VIABILITY

USING FLUORESCENT MARKERS TO ASSESS VIABILITY ALTERNATE

BASIC [3H]THYMIDINE UPTAKE TO ASSESS CELL GROWTH

BASIC CELL CYCLE ANALYSIS WITH PROPIDIUM IODIDE TO ASSESS CELL

MTT DYE CONVERSION TO ASSESS METABOLIC CAPABILITY BASIC

BASIC ALAMAR BLUE REDUCTION TO ASSESS METABOLIC CAPABILITY

MEASURING FLUORESCENT DYE LOSS TO ASSESS METABOLIC BASIC

Dye extractor solution

Specific absorbance ± SEM

Test chemical and Percent of control (±SEM) atc

Application notes for the Cytofluor Fluorescent

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