Hello everyone,
I would like to study human PBMC mRNA gene expression for some receptors after culture with antipsychotics drugs. I have some doubts.
How long should I culture PBMCs with those medicaments?
In which way can I separate PBMCs from supernatant in 48-well plate?
Good day,
Some general advice: first of all, check if there is literature available on the experiment you say so you use it as starting point. Regardless, usually PBMC need some kind of stimulant (LPS, PHA, CpG, etc) in order to proliferate/activate, the incubation time should be optimized depending on the stimulant used and the molecule you want to measure. I have done this in different intervals: 2 h for evaluating oxidative burst, 24 h for cytokines and 72 h for cell subset evaluation.
I recommend you run a cell viability assay PBMC + drug concentration in order to validate any result you might get (of gene expression) in terms of concentration of the drug used and incubation time.
Remember PBMC include adherent (monocytes) and non adherent (lymphocytes) cells, so for cell/supernatant separation, I highly recommend you centrifuge the plate directly pipette supernatant out and do the lysis step on the plate.
It is not much but I hope it helps.
Good luck
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