Culturing the cells

Make media

Dmem 1 bottle + 10% Fbs(-20 fridge) + 1% antibiotic – Pen

Strep(-20 fridge)
For 500 ml Dmem bottle, we add 50 ml of Fbs( fetel bovine
Serum) + 5 ml of antibiotic( penicillin streptomycin).

Thawing of the cells

1) Put 5 ml of media in each tube depending on number of

batches of cells you are thawing.

2) After you take out the cells from -80. C/liquid nitrogen, thaw
them in the water bath for a minute or less until it's liquid.

3) Put the sample in the tubes as labelled.

4) Centrifuge (balance with equal volume tubes)at 1000 RPM for

5 minutes.

5) Aspirate (make sure you do not suck up the cells)

6) Add media to the plate or flask depending upon what you are
using for culturing the cancer cells.
 For a 6cm plate, total volume of media needed = 5 ml
For a T25 flask, total volume of media needed = 10 ml
For a T75 flask, total volume of media needed = 23 ml

7) Accordingly add media to the tube containing the cancer cell

pellet.
Re-suspend it multiple times.
Add cell suspension in media to the plate/ flask (uniformly
distribution)

8) Incubate at 37. C

Passaging

1) Aspirate the media

2) Wash with PBS (7ml so that cells do not dry out and makes
trypsinizing process quicker)

3) Aspirate the PBS

4) Add Trypsin (.5% or .25%)

Trypsin amounts for different plates are as follows

T75 cm flask. 3 ml
T25 cm flask 2 ml

10 cm plate. 3 ml

6 cm plate. 2 ml

35 mm plate 1 ml

5) After adding Trypsin, incubate for 6-7 minutes depending upon

the cancer cells.
Hsc cancer cells take about 6-7 minutes to detach whereas 14 a
cancer cells take 9-10 minutes. Also if cells are being cultured on
a plate for long time, .25 % Trypsin is required.

6) After incubation, check for the floating (detached) cells in the

plate under the microscope.
7) If some are still attached while the majority is floating, gently
tap the sides of the plate to detach the remaining cells. If the
majority of the cells are still attached, keep it back in the incubator
for a couple of minutes and check again.

8) After all the cells are detached, transfer the content to the tube
containing media volume ≥ added amount of Trypsin.

9) Centrifuge at 1000 rpm for 5 minutes.

10) Aspirate
11) Add media to the cells

12) Add media containing the cells to the plate/flask

13) Incubate at 37.c