Cell Culture Protocol

Remember – Everything inside is clean, everything outside is dirty.

Reanimating Cells From Cryogenic Preservation

1. Prepare in culture cabinet, 50 ml of RPMI at 4C in falcon tube. Also prepare 50 ml of

2. Take cryovial of cells out of liquid nitrogen, place on side and let it warm up for 15 seconds.
Note, you will have 1 million cells if the cryovial contained cells from one T25 flask (A flask
which has capacity of 25 cm2 surface area). You will have 3 million cells if the cryovial
contained cells from one T75 flask (A flask which has a capacity of 75 cm2 surface area)

3. Warm up cryovial in water bath at 37ºC. When you see the last slither of ice turn into liquid in
the vial, take it to the cabinet.

4. Using a 1ml pipette, aspirate and dispense entire contents of cryovial (cells in fetal calf serum
(FCS)) three times to resuspend cells. Then aspirate entire contents (should be 1ml) and in a
drop wise manner, slowly add cells to RPMI.

5. Following this, take about 1 ml of the RPMI (+cells) and add it into the cryovial. Aspirate
contents and put it back into the falcon tube containing RPMI+cells. This is so that you ensure
removal of any residual cells left over in the cryovial.

6. Centrifuge contents of falcon tube at 800 ×g for 5 minutes

7. Following this, you should have a cell pellet at the bottom of the tube, take out RPMI media
and resuspended cells in 6ml media.

8. If the cryovial contained 1million cells (ie. one T25 flask worth of cells was frozen down)….

Put 8 ml media into two T25 flasks and add 2 ml cells (using the cells in 6ml media from part
7 of procedure above). Put into incubator. This is called sub-culturing with a cell density of 1
in 3. This density will require you to culture cells TWICE a week (eg. Monday and Thursday).

Alternatively, put 9 ml media into two T25 flask and add 1 ml cells (using the cells in 6ml
media from part 7 of procedure above). Put into incubator. This is called sub-culturing with a
cell density of 1 in 6. This density will require you to culture cells ONCE a week (eg. Every
Monday).

If the cryovial contained 3million cells (ie. one T75 flask worth of cells was frozen down)
….then just divide the amount added by 3. For example, For a 1 in 3 split = Put 9.6ml media
and 666 µl of cells.

Label the flask with following info: Your name, the passage number, date and cell density.

9. Put into incubator at 37ºC with 5% CO2.

Sub-Culture

1. Cells should be 75% confluent in the flask. Check this is the case before preparing media. If
cells are ready for sub-culture, defrost trypsin in water bath at 37C.

2. Prepare 50 ml cell media:

PNT2-C2 cells are cultured in RPMI with 1% L-Glutamine and 10% FCS.
 LNCaP cells are cultured in RPMI with 1% L-Glutamine, 10% FCS, 1% Sodium pyruvate and
1% HEPES buffer.

PC-3 cells are cultured in Hams F-12, with 1% L-Glutamine and 7 % FCS.

BPH cells are cultured in Keratinocyte-SFM media (supplemented with EGF and BPE) and
1% L-Glutamine.

3. Remove growth medium from flask and wash cells twice in PBS (10 ml for each wash). Then
add trypsin, the amount to add depends on which flask you are using, if you are using a T25,
add 1ml trypsin. If you are using a T75 flask, add 2 ml trypsin. In either case, after addition of
trypsin, put into incubator at 37 C for 1 minute. Following this, take cells out of the incubator
and firmly tap the side of the flask to loosen cells from the bottom of the flask. Check under
an optical microscope that the cells have detached from the flask bottom.

4. Promptly add 5ml respective culture media (if cells were originally trypsonised from a T25
flask) or 4 ml media (if cells were originally trypsonised from a T75 flask). The FCS
contained within the media will neutralise the effect of trypsin so that no further proteins will
be digested from the cell surface.

All cell lines we use media containing FCS, EXCEPT BPH. For BPH, you must add ≥ 10 ml
RPMI, then put into a falcon tube and centrifuge at 400 ×G for 5 minutes. Following this, you
should have a cell pellet at the bottom of the tube, remove RPMI and resuspended cells in
Keratinocyte-SFM media (supplemented with EGF and BPE) and 1% L-Glutamine.

You will now have 6ml cells in solution in the flask– aspirate and dispense three times against
the side of the flask so that you separate the cells apart as they like to stick to each other in
clumps. Then, put cells into a 15 ml capacity falcon tube.

5. Take two T25 or two T75 flask’s per cell line – the choice depends on how many cells you
require for your experiments. To start off with, we should use two T25 flasks / cell line.

9. Put 8 ml media into each T25 flask and add 2 ml cells (using the cells in 6ml media from part
7 of procedure above). Put into incubator. This is called sub-culturing with a cell density of 1
in 3. This density will require you to culture cells TWICE a week (eg. Monday and Thursday).

Alternatively, put 9 ml media into a T25 flask and add 1 ml cells (using the cells in 6ml media
from part 7 of procedure above). Put into incubator. This is called sub-culturing with a cell
density of 1 in 6. This density will require you to culture cells ONCE a week (eg. Every
Monday).

The choice of cell density depends on when you need the cells for experiments.

Label the flask with following info: Your name, the passage number, date and cell density

10. Put into incubator at 37ºC with 5% CO2.

Cells for Experiments

Section 1

For Paul and Caron, the following protocol is based on an experiment where 3 MirrIR slides are to be
plated with one cell line.

1. Sterilise MirrIR slides in 70% Ethanol in water for one hour in the culture cabinet and then
remove liquid and dry in cabinet. Remember to mark which side is the reflective side.

2. Put 3 MirrIR slides into a 6 well plate: one slide per well.
 3. Trypsinise cells off the flask and resuspended in 6ml media (follow parts 1 – 4 in “Sub-
culture”).

4. Take another falcon tube and add 13.2 ml media and 1.8 ml cells (you have added 3 × 10 5
cells). Shake well to mix cells. Then add 5ml media to each of the wells containing the MirrIR
slides (this should be 3 wells). You will have therefore added 1 × 105 cells to each well.

5. Place in incubator for 4 days at 37C with 5% CO2.

Section 2

For Paul, the following protocol is based on an experiment where 3 MirrIR slides are to be plated with
one cell line and stimulated with 50 uM deuterated palmitic acid (D31-PA).

1. Prepare 50µM D31-PA in ethanol. Remember D-PA is light sensitive.

RFM of D31-PA is 287

Take 0.01g D31-PA and dissolve in 1ml ethanol (Stock A). Stock A has a concentration of
34.84mM D31-PA (ie. 0.01 g / 287). *Remember 1000 µM = 1mM

Hence, 1ml of Stock A in 34.84 ml media will provide a concentration 1mM D31-PA.

To get 50µM D31-PA,

But,
We don’t really need 34.84 ml media for three wells – we really need 15 ml’s. So to avoid
wasting media we can scale down….
So, need 25 µl of Stock A in 17.42 ml media (in other words, 25µl stock A in 17.17 ml media)
to give 50µM D31-PA in media.

The media in this case will be RPMI with 1% L-Glutamine.

2. Follow parts 1-5 in section 1. However, after three days incubation – remove media from each
well and wash cells twice in PBS. Then add RPMI with 1% L-glutamine (pre-warmed to
37C). Continue culture for 24 hours (This is starving the cells).

3. Following starvation for 24 hours, replace media by washing cells twice in PBS and then
adding D31-PA containing media to each well (again 5ml / well). Incubate for 24 hour hours.
We know that at 24 hours we should see a strong C-D signal, but should start seeing C-D
signal even after 45 minutes incubation.

Fixation

Fix cells by removing growth medium, washing twice in PBS and then adding 5ml 4% formalin (in
PBS) to each well.

The following can be carried out on lab bench and does not have to be in the culture cabinet: Leave to
fix for 25 minutes and then remove slide and rinse in deionised water by dipping in three times with 2
seconds each dip. Then air-dry.
Time Requested

Practice Sub culture: 2 cell lines (PC-3 and LNCaP) to be sub-cultured by two experimenters. We
require two afternoons (1.00 – 3.00 pm) (Monday’s and Thursday’s) per week for three weeks. Starting
on 3rd December.

Experiments: We require 2 afternoons (1.00 – 4.00 pm) (Mondays and Thursdays) per week for two
weeks.

Consumables

This will be taken out of student grant codes (Please advise Sue).

We require two bottles of each of the following:

1. Nutrient Mixture F-12 Ham

2. L-Glutamine solution liquid, 200 mM, sterile-filtered, cell culture tested

3. Trypsin-EDTA solution
1 ×, 0.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks' Balanced Salt Solution with phenol
red., sterile-filtered, cell culture tested
£7.70
Cat no. T3924-100ML
Sigma-Aldrich UK

4. Keratinocyte-SFM Supplement
Human recombinant Epidermal Growth Factor (EGF 1-53) and Bovine Pituitary Extract (BPE).
Cat no. 37000-015
£24.00
Invitrogen

5. Keratinocyte-SFM (1X), liquid with L-Glutamine

I think Sue buys in their own stock of FCS, please check. I am not sure if PBS in supplied or not – if
not, ask Tim to order it in.

If you want to start culture as date specified, I suggest you start ordering in these media, but check with
elsa / tim to see what they already have in stock.