We have frozen PBMC isolated from human blood samples - (~3-10 million cells per patient).
We want to use them mostly for testing binding of various target molecules by BCRs and TCRs.
This means we don't mind some phenotype change and altered reactivity, so long as the cells survive and the BCR/ TCR repertoires are maintained.
Does anyone know of a protocol for expending T and B cells together out of a primary PBMC sample?
HI Gil,
For human T cell expansion anti-CD3/anti-CD28 + IL-2 stimulation works quite well.
I don't have experience with huB-cell expansion but for mice, anti-IgM, CD40L and LPS are routinely used.
I'd suggest you do separate wells for expanding T- and B-cells rather than together, there might be ways but it sounds a little "unconventional".
From 1-2million PBMCs you can get robust expansions (at least for T cells). I.e. one "hit" with anti-CD3/anti-CD28 would keep them expansing for ~2 weeks, yielding a 10-20 fold expansion. You could even re-stimulate again to keep them expanding.
Having said this, I'm sorry but I don't know this numbers for B cell.
I'm confident you'd be able to find good protocol online with the keywords above but let me know if you need more help.
I agree with Sergio, I think it'd be better to split your PBMCs into two and do separate T and B cell expansions, otherwise things could get messy.
Hello Gil,
I think you can also use various mitogens for expansion of T cells such as PHA (phytohemagglutinin) or Con A (concanavalin A), whereas for B cells for example PWM (Pokeweed mitogen). They are very efficient as I know from my practise. They are also usefull if you have patients' PBMC with antigen-specific T or B cells and you want only to perform clonal expansion using blast transformation test.
But if you want to intensify the proliferation of naive lymphocytes in response to antigen of your interest, the stimulants aforementioned by Sergio are needed.
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