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Clonogenic Assays Protocols

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36 views2 pages

Clonogenic Assays Protocols

Uploaded by

apvargas
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© © All Rights Reserved
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Clonogenic Assays Protocols

Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the
clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can
often only be determined microscopically. A clonogenic assay is the method of choice to
determine cell reproductive death after treatment with ionizing radiation, but can also be used
to determine the effectiveness of other cytotoxic agents.

Crystal Violet Assay

Materials and Reagents

 Cell culture medium (DMEM 10% FCS)


 Phosphate buffered saline (PBS)
 Trypsin/ EDTA
 Crystal violet
 Fixation solution
 Cell culture petri dishes or six-well plates
 Hemocytometer
 Stereomicroscope (e.g., Nikon Eclipse, model: TS100 )
 CO2 Incubator

Cell preparation:
1. Culture CF41 and MeLn cells according to their requirements.
2. Remove medium, and then rinse cells with 10 ml PBS.
3. Add 4 ml 0.25% trypsin to the cells and incubate for 1-5 min until the cells appear round
and detach from the substrate.
4. Add 10 ml medium with 10% FBS, and detach the cells by pipetting.
5. Count the cells using a hemocytometer.
6. Note: It is critical to get a relatively accurate number for the cells.
7. Prepare desired seeding concentration: 1x103 to 4x104 cells per milliliter of complete
culture medium, and then seed cell into 6mm dishes.

Assay setup:
1. Incubate cells for a few hours to overnight in a CO2 incubator at 37 °C and allow them
to attach to the plate/dish.
2. Treat the cells as necessary with chemicals, radiation or a combination of both.
3. Incubate the cells in a CO2 incubator at 37 °C for 1-2 weeks until cells in control plates
have formed colonies that are of a substantially good size (50 cells per colony is the
minimum for scoring).
Fixation and staining:
1. Remove medium, and then rinse cells with 10 ml PBS.
2. Remove PBS and add 2-3 ml of fixation solution and leave the dishes/plates at room
temperature (RT) for 5 min.
3. Remove fixation solution.
4. Add 0.5% crystal violet solution and incubate at RT for 2 h.
5. Remove crystal violet carefully and wash the dishes/plates in distilled water to rinse off
crystal violet.
6. Air-dry the dishes/plates on a table cloth at RT for up to a few days.

Data analysis:
1. Count number of colonies with a stereomicroscope.
2. Calculate plating efficiency (PE) and surviving fraction (SF).
3. PE = no. of colonies formed/ no. of cells seeded x 100%
4. SF = no. of colonies formed after treatment/ no. of cells seeded x PE

Soft agar assay (Clonogenic anchorage-independent assay)

1. Prepare single-cell suspensions according to the Cell Preparation item up to step 6;


2. Mix 1x103 to 4x104 cells in 1mL of 0.6% agarose prepared in complete medium;
3. Plate cell-agarose mixes in culture dishes coated with 5mm of 1% agarose (already
prepared by your instructors);
4. Incubate plates at 37 °C and 5% CO2 for 2 weeks to allow colony formation;
5. The number of colonies formed and the colony area (mm2) are determined under the
light microscope;
6. Analyze using ImageJ software (NIH, Bethesda, MD, USA).

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