Cell Lines and Culture Medium (All Medium in TC Lab Fridge)

Cell lines and culture medium (all medium in TC lab fridge):

1. MCF-7-these are to be cultured in DMEM (complete) 2. SKBR3-these are to be cultured in McCoy’s (complete) 3. 184B5-these are culture in MEBM

184B5-change the medium on these cells by pipetting off old medium and adding 5ml for t25 or 15ml for t75 of new medium (pouring off medium encourages contamination by wetting the neck of the flask.)

MCF-7 and SKBR3 will probably require subculturing:

1. pipette off medium 2. wash with sterile PBS (on top of fridge-pour off) 3. add 1ml of 10x trypsin-EDTA (thawed stuff in fridge door or 10ml aliquot in freezer of fridge in tissue culture lab). 4. place at 37C (only for about 1min)-cell should start lifting off-give the flask a few brisk taps and check that all the cells are lifting off. 5. Add 10ml of the appropriate pre-warmed complete growth medium to the tryspinised cells and transfer to a 15ml Falcon tube. Spin down cells, 5min, 2000rpm, RT. 6. Tip off the medium. Replace cap and break up cell pellet by violently flicking the bottom of the tube. 7. Transfer 1:3 (if confluent) to a new t75, i.e. add 14ml of growth medium to flask and resuspend cells in 3ml and add 1ml of this to the new flask. 8. Please keep track of passage number: in the form 2.4 would then be 2.5 etc.

VLB100 cells

When they reach 1 x 106 cells/ml then transfer them into a t175cm flask (should be some opened in the ‘in use’ bin), in a volume of 50ml.

Then, when they are approaching subculturing density, spin down enough cells for 5 aliquots of (1 x 107 cells).

1. Prepare freezing medium: 60% growth medium, 30% FBS (should be a smaller than 50ml aliquot in freezer in TC lab), 10% DMSO (sterile on the bench inTC lab). Mix, then filter sterilise into 15ml Falcon and cool on ice. 2. Resuspend the cells (after breaking up pellet by flicking into 5ml of the chilled freezing medium and aliquot into appropriately labelled 2ml screw cap tubes (some open). 3. Place in Mr Frosty (may want to replace isopropanol first) and place at -70C 4. Transfer the cells the following morning into box 1A of the cryostore, recording the positions within the box to make it quicker/easier to find in future. 5. Use remainder of cell culture to subculture and this can now be utilised as a working stock for about another 10 passages.