Cell Transformation Group:Protocols/Cell Culture

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'TO FREEZE:'

1. Lift cells when 70 – 80% confluent and resuspend in 10% FCS/DMEM (~8ml) (80 cm2 FLASK⇨ 1 X 107Cells/ml).

2. Spin at 250g for 5min. Discard S/N. Resuspend in 2ml of DMEM (or 4ml for large flasks). Put on ice.

3. Mix 12ml DMEM (+10%FCS+PenStrep) with 4ml FCS and 4ml DMSO (add DMSO [flammable goods cupboard] last as a precipitation forms) and chill on ice. This is the freezing mix. IMPORTANT - If you are only freezing one or two cell lines down, then adjust the final volume of freezing mix so that there isn’t so much waste (need approx 2ml mix per med flask and approx 4ml for a large flask).

4. mix at a ratio of 1:1 the freezing mix and cell mix, by adding dropwise the freezing mix to the cells. gently shaking to ensure mixing.

5. 1ml of mix to each cryovial on ice. transfer to “Mr Frosty” and freeze at -70°C for 24hrs. Make sure to mark off Mr Frosty usage by crossing out number on lid. Refill with isopropanol after 5 usages.

6. transfer to liquid nitrogen and add to cell lines log book.

'TO THAW:'

Speed is essential for the first few steps as DMSO is harmful to cells and the less time they are in contact with it the better.

1. Remove cells from liquid nitrogen. Thaw quickly at 37°C.

2. Pipette into warm complete DMEM (10ml).

3. Spin at 250g for 10 mins.

4. Resuspend cell pellet in 5ml of DMEM and transfer to medium flask (80 cm2)† containing 20ml of DMEM.

5. Incubate in 37°C CO2 incubator. Delete from cell lines log book.

If Thawing Lots of Samples (>5)

1. Fill chilly bin with dry ice from -70°C freezer

2. Remove cells from liq N2 and place on dry ice

3. Place chilly bin (with cells) in -70°C

4. Pre-label a medium flask† and a plastic universal for each cell line being brought up. Add appropriate amounts of DMEM to each

5. Take 4 (or appropriate number) vials out at a time and thaw at 37°C

6. Pipette entire contents of thawed vial into its corresponding universal

7. Spin at 250g for 10min [removes DMSO]

8. Use a 10ml pipette to resuspend the cell pellet in 5ml DMEM and transfer suspension to a medium flask

9. Incubate at 37°C. Delete from cell lines log book.