I have been running ELISPOTS with not much success recently due to high levels of background. After testing everything from the DMSO used in peptide stimulation to the FBS in the culturing medium, I fear that the high background is attributable to apoptotic cell death. I fear that I may not be working quickly enough. During the experiment, there is typically a 1-2 hour window where PBMCs are outside of the incubator in R-10 culturing medium. Is this enough time to cause significant cell death?
Hi Philip, please check bellow site:
https://blog.quartzy.com/2017/05/30/peripheral-blood-mononuclear-cells-pbmc-isolation-preservation-culture
Dear Philip Samaan,
Yes, we also faced similar problems. From our experience, I would request you to make two modifications:
1. Please use Ficoll-paque premium density gradient media (GE Healthcare) for isolation of lymphocytes. As this special medium contains very low levels of endotoxin (< 0.12 EU/mL), you will be able to get > 90% viability of the separated cells.
2. For brief incubation/culture, please use LGM-3TM Lymphocyte Growth Medium (Lonza) which is a chemically defined, serum-free, xeno-free complete media containing human albumin, recombinant human insulin, and pasteurized human transferrin.
Hope this helps.
Best wishes & regards.
I think 1-2 hours outside the incubator is too much time for the cells to bear. The two important criteria are temperature maintenance (37 deg C) and pH maintenance of the media (by CO2 input) both of which are not met when the cells are outside the incubator
Cell death due to these factors can not be ruled out
Best,
- Badges
- Science topic