Isolation of adherent cells from TC flasks. FGB

Revised 8/1/96

Reagents: Media waste container in TC hood, aspirator, Trypsin, PBS, Conical tubes, 4'C Centrifuge, Dulbeco's Modified Eagle Medium 3+ ( 50ml HI-FBS, 5ml Pen.-Strep. and 5ml L-Glutamine).

Note: All steps are to be performed aseptically at all times.

Note: Use DMEM+3 for pig cells and RPMI +4 for T-cells.

1. View to verify confluent growth under microscope.

2. First empty TC flasks.

3. Add 20ml (to 185ml TC flasks) of PBS to rinse FBS.

4. Sit flat 5 minutes then repeat.

5. Empty again.

6. Thaw Trypsin in 37'C water bath then put on ice. Mix, add 4ml Trypsin.

7. Sit flat 3 minutes. Then bang cells loose. Verify under scope.

8. Add 10ml media or incubate 37'C to stop trypsin, rotate flask w/out getting cap wet!

9. Stand upright, then pipet into a 50ml conical tube after rinsing from 3/4 up 3x.

10. Pool if possible.

11. Spin at 1200 rpm/4'C/10 minutes.

12. Decant off the media.

13. Spin cells at 1000 rpm/4'C/10 minutes then aspirate off the media.

14. Add 10ml to pellet then gently redissolve the pellet. Combine all tubes if applicable.

15. To freeze cells, see “freezing cells” protocol. To use cells, resuspend in 10-20ml media, depending on the cell concentration.