Hello immunology experts!
I am working with human PBMC's and doing T cell studies. Wondering what is the typical percentage of cells that you lose after overnight resting before stimulation.
Thanks!
Krupa
Hi Krupa...
Please clarify, how will you rest the cells ? At what temperature and the suspended medium...? So that you would be clarified.
Usually, the isolated PBMCs were suspended in FBS+DMSO and stored at frozen state by following gradual freezing method. By this way you can rest the cells for overnight or longtime.
And the another important thing is Thawing of frozen cells before stimulation by following slow thawing. This performance will reflect on the percentage of viability. So, the thawing after overnight would have 90 to 95 percentage of viable cells if you processesed it carefully.
Now find where the problem begins in your protocol.
Cheers,
Kadar Moideen Abbas
We store PBMCs at 2x10e6 per ml overnight at 4C in RPMI 10% FBS with miminal loss of T cells. You will lose some monocytes which have a tendency to adhere to plastic. We also enrich CD4 T cells using MACS before keeping them resting.at 2x10e6 in RPMI 10% FBS at 37C up to 5 days. You can then activate them and they will be fine.
Thanks for your responses. These are thawed PBMC's after storage in freezer for a few months. I have them resting in R 10 media overnight in a 37 degree CO2 chamber but had about 30% cell loss the next morning. I used 15 ml conical tubes with lose cap to rest them. Any suggestions??
I suppose that the temperature was too high. We store our PBMC's at 4°C overnight in 20 % FCS and 10 mM EDTA (in PBS) in Falcon tubes and do not measure such a high cell loss. I think you can not handle them like an immortalized cell line. Good luck for your experiments.
Maybe the problem is with freezing method. In my opinion the freezing must be slow (cooling very close to -1°C/minute) and the thawing of the cells must be very fast, since this prevents the formation of crystals and the DMSO toxicity.
After thawing with 4h in medium with 20% DMSO is enough for a good viability
Try using 50 mL conical tubes, re suspend in R10 at a concentration of 1x10^6 per mL and tilt the tube at a 45 degree angle
I would recommend referal to:
Smith, JG, et al 2007, Establishing acceptance criteria for cell-mediated immunity assays using frozen peripheral blood mononuclear cells sorted under optimal and sub optimal conditions. Clin Vaccine Immunol.. 14:527-537.
.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes