Recently, I've been investigating B cells generated by my vaccine, I have found some great results, but now I would like to quantify specific B cells using flow cytometry and stain the specific B cell for sorting. I would really appreciate with someone would have a protocol or a comercial kit to recommend.
Hey Abdullah Al Tarique
, thanks for answering. I get that, I already use this panel to stain B cell, the problem is that using that markers I can't quantify specific B cells, the cells generated by my vaccine. I was looking for a kit or protocol that conjugate a recombinant protein with fluorochrome, by that I can stain B cell that recognize my protein.
Antigen-specific B cells in blood of humans are of two types.
Plasmablasts that secrete antibodies and will become plasmacells after homing to tissues and memory B cells that do not. Using ELISPOT (as suggested above by Abdullah Al Tarique) is a good method to detect antigen-specific plasmablasts. If you need to detect memory cells you either need to use specific antigens coupled to fluorochromes for staining (and this can bit tricky with background) or you can stimulate the memory cells to produce antibodies (for example using stimulation with R848 and IL2 as originally described by the group of Antonio Lanzavecchi in DOI 10.1002/eji.200839129) and then determine the presence of cells using ELISPOT or Elisa.
Thank you Mats Bemark , but i already did ELISPOT for different sites and different time points so the kinetics of the B cells, right now I want to do single cell rnaseq to answer other questions. thanks again for the protocol or stimulating memory B cell I was also looking for that.
Dear Isau Noronha ,
It's quite a complex task to identify the B cells generated only in response of your vaccine. Frankly speaking, depending upon the route of vaccination, B cells response could be different. It's unlikely to find a new set of specific B cells generated against your vaccine. Even if they generate, you have to identify their origin first. Percentage of B cells in bone marrow & spleen of mouse model significantly differs. If your mice were healthy, there will be less of new B cells generation , moreover B cells in ontogenetic stages will respond through multiple mechanism including antibody production against your vaccine. I think, for your study, you need to first study the vaccines response of B cells in vitro ( sort & study ). Or, you can classify each subsets of B cells ( from Pro, pre, mature, plasmablast etcs )and try to distinguish their response.
Feel free to ask for further discussion.
Best regards,
Babu M.
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