I need to separate B lymphocytes from blood and maintain a cell line of it. Regarding that how should I proceed with B cell separtion from whole blood of a teleost fish of Cyprinidae family. For that I need to find out certain B cell surface specific markers based on which I could go for cell sorting. If anyone could suggest me B cell surface markers specific to carp family and from where can I procure it (under which brand), it will be helpful. Through literature, I do know certain carp Immunoglobulin positive B lymphocyte markers but I am not sure, whether those would work in my case and secondly, from where should I order.
Hi,
Check this paper
http://www.ncbi.nlm.nih.gov/pubmed/14659910
Regards
Akshay
I think if you use native peripheral blood from carp, you'll have several difficulties. You may find a purer source of lymphoid cells in the pronephros (head kidney), but I'll leave this up to you. The first difficulty in using peripheral blood will be to isolate the buffy coat of mononuclear cells readily. Unlike mammals, in lower vertebrates, the buffy coat is often not nearly pure lymphocytes, platelets and monocytes, but rather contains many different cell types. The second difficulty will be identifying B cells and separating them from the other cell types. The third will be to develop and maintain a cell line of B cells isolated from carp blood in the lab, which may be very difficult indeed. Good luck with all these things! JPCaruso, PhD
Hello Akshay and Joseph. Akshay thanks for the paper. Joseph you are right regarding the difficulties of the work, but I am interested in separation of B cells from other cell types by exploiting carp B cell surface specific markers through FACS. Now, the problem is I do not have much idea regarding what markers I should use for this work and where from I could order them.
Since you don't know which markers to use, you may want to look for a anti-IgM vs. this or other teleost species, as long as it's mu chain-specific. This would work to target naive (i.e. unstimulated) B cells. You may also care to check out recent work on IgD in lower vertebrates. Some investigators (Flajnik and others) have shown IgD in many fishes. If you want to assay total Ig-bearing cells, you could also look for an antibody that is heavy-chain specific vs. IgY (or its equivalent) in teleost fishes. I'm suggesting you use anti-Ig antibodies, because these usually cross-react with all members of a given lower vertebrate group. I've had to make my own antisera to work with reptiles, but I've also found evidence suggesting cross-reactivity with other species in the group. If you're looking to find anti-CD molecules as is common in conventional Immunology, I have no idea whether specific B cell markers have been described in teleosts. If they have, you could try one of these. Good luck! JPC
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