TITLE: Passaging adherent cancer cell lines
AUTHORS: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Fiona Behan, Rizwan Ansari, Mathew Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP is for the routine maintenance and expansion of adherent cancer cell lines. </div><div class = "text-block">It also details how cells are harvested and counted for use in downstream protocols.</div><div class = "text-block">This protocol is also used in expasion of Cas9 transduced cell lines, prior to banking.</div><div class = "text-block">Process diagram:</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/b94e6458af3e4aa17dc6c162a5d28b87f529d83e9d05c0764f6eb3fffa72f21d/ccgfbdsax.png" /></div></div>

[STEPS]
?. [Detaching and collecting cells]
Remove the old culture media using a sterile aspirator pipette.
?. [Detaching and collecting cells]
Gently wash the cells with PBS (see Table 1, column B for recommended volume). Aspirate the PBS from the flask.  ABCD1Size of flaskVolume of PBS (ml)Volume of TrypLE (ml)Volume of media and cells (ml)2T255373T757.54124T150105245T525 (3 layer)3015606T875 (5 layer)5025100
Always aim to add reagents at the side/back of the flask initially to avoid dislodging any cells.
ABCD1Size of flaskVolume of PBS (ml)Volume of TrypLE (ml)Volume of media and cells (ml)2T255373T757.54124T150105245T525 (3 layer)3015606T875 (5 layer)5025100
?. [Detaching and collecting cells]
Add sufficient TrypLE Express Enzyme to cover the bottom of the flask (see Table 1, column C for recommended volume). Rock the flask to allow the TrypLE to cover the surface of the cells.
?. [Detaching and collecting cells]
Incubate the flask at   for 3-5 minutes. Use the microscope to check if the cells have detached. If necessary, tap the side of the flask, or return to incubator until all cells have detached.
37 °C
?. [Detaching and collecting cells]
Add fresh media to the flask at an equal volume to the TrypLE added in Step 3. Use the liquid in the flask to rinse the cells off the bottom of the flask. Transfer the entire volume to an appropriately sized sterile tube.
?. [Detaching and collecting cells]
Spin the tube at  for 3 minutes.
Centrifuge: 300 34
Tubes should be spun inside centrifuge buckets with sealed safety caps. After spinning, the sealed bucket should be transferred to a microbiology safety cabinet before removing the tubes.
?. [Detaching and collecting cells]
Aspirate the supernatant, taking care to avoid disturbing the cell pellet. Resuspend the pellet in an appropriate volume of fresh media (around  - ) and mix well to ensure a single cell suspension.
5 mL
20 mL
?. [Detaching and collecting cells]
If a cell count is not required, proceed to Step 9. To take a cell count, aliquot  of cell suspension and count using your preferred method.
100 µl
?. [Re-seeding cells]
Add fresh media and an appropriate volume of cell suspension to a new flask, to achieve the desired split ratio or cell number (where possible, aim for the total volume of media and cells in the flask to follow the recommeded volumes in Table 1, column D). For example, if splitting a T75 flask 1:2, either add the entire cell suspension to fresh media in a T150 flask (achieving a total volume of  ), OR add half the cell suspension to fresh media in a T75 (achieving a total volume of  ).
24 mL
12 mL
The required split ratio will be dependend on the growth rate and behaviour of individual cell lines.
?. [Re-seeding cells]
If maintaining a selected population of Cas9 cells, add blasticidin to achieve the desired concentration, as determined by: 'Blasticidin titration of cancer cell lines' protocol. Add the required volume of 10mg/ml blasticidin depending on the flask size (Table 2), to achieve the desired final concentration.  ABCDEF1Size of flaskVolume of media (ml)10µg/ml Blasticidin (µl)25µg/ml Blasticidin (µl)50µg/ml Blasticidin (µl)75µg/ml Blasticidin (µl)2T257717.53552.53T7512123060904T1502424601201805T525 (3 layer)60601503004506T875 (5 layer)100100250500750
ABCDEF1Size of flaskVolume of media (ml)10µg/ml Blasticidin (µl)25µg/ml Blasticidin (µl)50µg/ml Blasticidin (µl)75µg/ml Blasticidin (µl)2T257717.53552.53T7512123060904T1502424601201805T525 (3 layer)60601503004506T875 (5 layer)100100250500750
Blasticidin is toxic if swallowed and harmful in contact with skin.
?. [Re-seeding cells]
Transfer the flask to a  , 5% CO2 incubator. Agitate the flask gently back and forth and side to side to ensure even distribution of cells across the flask.
37 °C
?. [Re-seeding cells]
Inspect the cells daily and record confluency. Cancer cell lines should have a complete media change twice per week, or as needed if the media looks yellow.