We will assess viral-antigen function in PBMCs after virus challenge.
There is discussion of adding human AB serum instead of FBS or autologous serum. Any comments, taking in mind factors in autologous serum that would support Tfh function and or B cell differentiation during viral challenge?
In our antigen-specific (but also non-Ag-specific) cultures, we use human AB serum to reduce aspecific responses - compared with FBS.
If you use autologous serum, you will use variable sera, dependent on the subjects. In that case, you have more than one variable (namely: serum & cells). I would reason that the use of one batch of human AB serum is preferable, to make sure you only look at cell-intrisinic properties (unless you are interested in the question what the serum does, but then you rather use one pool of cells to test several sera).
Thank you both for your suggestions.
Adriaan can you tell me the experimental details of your T-B cocultures, eg. days of culture, activation, number of cells, culture medium, plates etc.?
Thanks
We coculture electroporated autologous B cell blasts (14-21d IL-4/sCD40L culture from PBMC) with tumor-infiltrating leukocytes (includes also non-T cells) for 6-7 days. Typically, we use 2x10^5 B cell blasts, and 2x10^5 tumor-infiltrating leukocytes. 96-wells round-bottom plates. Medium: RPMI (with ultraglutamine), 10% AB serum, 1% Pen/Strep, 1% ITS, 1% NEAA, 1% sodium pyruvate.
With these cultures we identify antigen-specific T cell responses (class I and class II).
Important note, the B cell blasts are frozen, and thawed when tumor-infiltrating leukocytes become available.
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