I'm trying to stimulate Jurkat T cells using ionomycin and PMA for 2 hours. I'm following the protool used by Diehn et al 2002. Basically:
1. Grow Jurkats to 1.0 Mio/ml in RPMI + 10% FCS.
2. Spin at 300xg, 10 min.
3. Remove supernatant
4. Resuspend in RPMI with 1 uM ionomycin and 25 ng/ml PMA (to a density of 1 Mio/ml).
5. Incubate in 37C water bath
6. Halt stimulation with 2:5 dilution with ice-cold PBS
7. Spin at 1,100xg, 4C, 6 min to harvest.
However, when I try this, sometime during the 2-hour incubation (it seems to happen after 1 hr), my cells aggregate and form an insoluble "snot"-like clump. When I spin down to harvest, this pellet is practically insoluble in RIPA buffer, so I cannot lyse the cells.
What is going on? I've never seen this happen with anti-CD28/anti-CD3 costimulation before, and the protocol is practically the same procedure.
Also, can I do anything to recover these clumpy pellets?
Thanks a lot!
Edited by chuggleschuggles, 23 October 2014 - 01:24 PM.
