I have tried using anti IgM and LPS without IL4 in PBMCs, but I don't see an activation of B cells. What should my read out be for B cell activation in ELISA or ELISPOT?
Hi Behnaz.
I would recommend using the protocol described by Pinna et.al 2009. This protocol uses 1µg/ml R848 and 10ng/ml IL-2 (or IL-21) and 72hr stimulation. With stimulation 72hrs and this protocol you get a very strong activation of B cells (both total IgG and antigen specific).
With 72hr stimulation, you also get higher viability of the B-cells, and you are able to perform more tests/week compared to other 120hr stimulation.
Another advantage to the CpG-based stimulation is that R848 is synthetic which means that there is very low batch variation compared to CpG.
You can read about our comparison of B cell activation protocols here:
http://www.ncbi.nlm.nih.gov/pubmed/23454005
Please let me know if you have any questions.
If you would like to activate B cells to measure IgG in ELISA or count the total IgG or antigen specific IgG secreting cells in ELISPOT assay, you need to stimulate them for a few days. There are a lot of protocols using different reagent for this aim. It actually depends on your PBMC species. Are they human or animal PBMC?
thanks for your prompt response. they are human PBMCS. i have found some protocol where they stimulate cells up to 5 days using PWM,IL4 and other stimulants. Which protocol you are referring to? appreciate your help.
These papers can be helpful to get idea:
http://www.jimmunol.org/content/183/4/2708.full
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000770
You can stimulate your PBMC with Pokeweed, CPG and IL15/ SAC.
Just keep in mind that because these methods stimulate total IgG memory B cells, so it is not very critical which method you select for stimulation. Just follow one paper protocol and modify it according to your aim.
Hello
I would recommend also this paper by Cao et al
http://dx.doi.org/10.1016/j.jim.2010.03.009
They compared various stimulation methods in an ELISPOT assay
Hi Inga,
If you are asking regarding stimulation of pure B cells, yes it has worked for me. I stimulated only pure memory B cells with IL-10, 1L-2, PWM and anti-CD40 and worked nicely. I could detect memory IgG B cells in ELISPOT and IgG secretion in ELISA.
Hi Behnaz.
I would recommend using the protocol described by Pinna et.al 2009. This protocol uses 1µg/ml R848 and 10ng/ml IL-2 (or IL-21) and 72hr stimulation. With stimulation 72hrs and this protocol you get a very strong activation of B cells (both total IgG and antigen specific).
With 72hr stimulation, you also get higher viability of the B-cells, and you are able to perform more tests/week compared to other 120hr stimulation.
Another advantage to the CpG-based stimulation is that R848 is synthetic which means that there is very low batch variation compared to CpG.
You can read about our comparison of B cell activation protocols here:
http://www.ncbi.nlm.nih.gov/pubmed/23454005
Please let me know if you have any questions.
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