What would happen to PMBCs When we isolate PBMCs from blood from a healthy person, and culture them in complete medium (RPMI1640+10%FBS+PanStrap) without any stimulation?
My questions are;
1. Will PBMCs lie, divide and maintain their counts or will die after some time without stimulation?
2. If PBMCs will proliferate, which subset of PBMCs would be proliferating much (T, B, NK or Mono)?
2. Do primary monocytes proliferate in cell culture, if yes what proliferating agent s to be used?
This is what we are planning, it is part of our research to isolate PBMCs and culture it in RPMI Gibco, we are to apply certain compounds on it, whether they show cytotoxicity in PBMCs or not.
This is what we are planning, it is part of our research to isolate PBMCs and culture it in RPMI Gibco, we are to apply certain compounds on it, whether they show cytotoxicity in PBMCs or not.
This is what i have observed:
1) Without any stimuli, monocytes will attach a few hours later, but their dendritic are short and keep intact for a few days, lymphocytes keep alive for 2-3 days, then turn senescence, will die around 5-7 days;
2) low cytokines (such as 1ng/ml IL-2) and refresh half medium every 2 days, lymphocytes keep alive even in day7, but they dont proliferate, nor secret cytokines. Strikingly, monocytes spread out and turn big and irregular, look like fibroblast cells.
3) Co-culutre with CD3 Abs or CD3/CD28 Dyna beads. T cells grow rigorously, turn big and more granuars, and produce different cytokines from different subsets, no obvious monocytes were observed.
4)If PMA/PHA/Ionomycin applied as stimuli, lymphocytes will turn into lymphoblast in a few hours, producing cytokines, and then most will execute apoptosis one day later.
If cultured in PromoCell's Mononuclear Cell Medium, the mixed population will stay unchanged for about 48 hours. But then the ratio of cell types will change - some sub-populations proliferate, some differentiate and some die.
Agree with Shiqiu. I use 1640+10%FBS+100u/mL IL-2 to culture thawed PBMC. After 2 days culture than anti-CD3/CD28 for 72h, there will be 30~40% lymphocytes grow like "balls". If not treated with anti-cd3/cd28, that will be 10%, and not so large(the total cell counts may not increase).
I think T cell can still proliferate after 7days(2d+3d+3d coculture), but they might be not so cytotoxic
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