Passing 293 T cells

 

1. Take plates, suck of media using blue tips and suction

2. Wash with HBSS (approx. 5 ml/plate) to remove FCS (allows trypsin to work)- careful to add solution by tipping the culture plate so as not to disturb cells attached to the bottom

3. Suck off HBSS again with blue tips

4. Add trypsin and swirl plates to dislodge cells from the bottom (leave long enough to see monolayer removed from surface, but not to damage cells)

5. Tilt plates forward and remove some media and cells from plate, rinse plate from top and collect again at the bottom of the plate. Put into a tube. (Careful not to allow too much bubbling).

6. Add approx. equal volume of DMEM to the tube, swirl.

7. Pellet the tube in centrifuge at 1000 rpm for between 5-10 minutes.

8. Pour off supernatant completely into another waste tube

9. Resuspend pellet in DMEM (enough for a 1/10 dilution).

i.e.: Add 10 ml to resuspend pellet, add 1 ml to each culture plate, then add approx. 5-9 ml more of media to cover bottom of plate