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Stimulating Jurkat T Cells with PMA: Clumping Cells?

jurkat stimulation pma clumping

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#1 chuggleschuggles

chuggleschuggles

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Posted Yesterday, 01:08 PM

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, Yesterday, 01:24 PM.






Also tagged with one or more of these keywords: jurkat, stimulation, pma, clumping

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