Hi there, just wondering if anyone has the same issue, and if there is something you do, or just something that is accepted. I am performing a PBMC-PHA stimulated assay in 96 well plates. this is 100,000 cells per well, with a final PHA concentration of 10ug/ml. i then analyse via flow on day 4. I am finding as time goes on, that the media of the well containing PHA turns yellow (i.e media exhaustion, pH change). is this something that is suppose to occur? does anyone change media during the assay? if so how?
also, with aggregates that form with PHA, i am now trying to use EDTA to break these up so i can analyse, but im still getting aggregates/lumps of PBMCs even after EDTA, is this too ok? how do others analyse via flow, and whats your protocol, and what kind of numbers of PBMCs are you getting after PHA addition for 4 days?
thanks in advance!
Media color change is normal as T lymphocytes are activated and proliferate during those 4 days. For that amount of time, I wouldn't change the medium. If you have to incubate longer, you could spin the plate, remove half of the media using a multichannel pipette (being careful) and put back the removed volume of fresh media.
Clumps and aggregates are also normal and you have to use at least 5 mM EDTA in PBS for 15 min with many pps and downs using a pipette to disperse the cells prior to flow cytometry. Keep 2 mM EDTA in your staining and washing buffers. Even with 5mM EDTA, you could still observe clumps, activated cells are very sticky (they express a lot of adhesion molecules).
Good luck,
thank you so much!!! this answered everything...and very reassuring im on the right track with everything! thank you :)
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