Hi guys! I'm planning on performing a crude isolation of B cells from PBMCs by nylon wool before I stimulate them with CD40L, IL-4 and IL-10 (to induce production of all Ig subtypes). The majority of PBMCs will be stored in liquid nitrogen prior to this. Should I culture these cells overnight after thawing, to relax them before I separate them on nylon wool? I don't want to stress them out and have them die.
If so, do I need to coat the culture plates with autologous plasma beforehand? I don't readily have access to corresponding plasma for a lot of these cells...
I would not recommend to "rest" mouse B cells before separation. 30% of them would die in 24 hours in culture even if taken fresh, not frozen. Human B cell are probably not that fragile, But still I would rather do all the procedures as soon as possible.
I agree with Natalie.
It is very important to activate B cells as soon after thawing.
Anyway 50% of the cells will die, despite activated via CD40.
The activation by CD40 does not require the addition of cytokine for all isotypes, if you work from peripheral blood. All isotypes are present naturally.
If working with naive B cells extracted from tonsil, a cytokine cocktail is necessary to switch isotypes: Il2, IL4, IL10 ...
Be careful, if you add human plasma in your culture, you can not quantify the different isotypes are looking to follow.
Anyway CD40 system is so powerful that it is sufficient unto itself
Thank you everyone for your prompt responses. Natalie, I am working with human B cells, it's good to know that they're a bit more robust.
So I'll isolate them immediately after thawing, and then immediately culture and stimulate. I won't be quantifying supernatant Ig levels unless absolutely necessary (ELISA is expensive). I'm going to be looking at AID mRNA, switch circle transcripts, Ig germline transcripts and productive transcripts by reverse transcription PCR. I still need to work out when to harvest the cells as well... but I can do that a little bit later.
Hi David,
I would recommend to culture the PBMCs overnight (usually RPMI+10%FCS) since a lot of receptors get downregulated when you freeze the cells, which could be problem for your seperation approach. That should also beneficial for your yield. Anyway never worked with B-cells, though but thawing PBMCs and cell loss was never a problem. Just make sure that you get rid of the DMSO asap.
Ingo
I would compare with and without the overnight incubation in medium/ FCS. Some people dilute out the DMSO after thawing and some pellet the cells. You should compare this too. Different labs do different things so you need to optimize the assay in your own lab.
It is interesting question. Sometime, I have no time to do all the procedures as soon as possible. I agree with Ingo Fricke`s opinion. The best way is to use fresh B cells to culture. If you thaw human PBMCs.it`s better to culture the PBMCs 1-2 fays with RPMI+10%human FCS. I don`t thisnk it is useful to precoating autologous plasma.
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