I am looking for cheaper and still efficient state of art methods. I have checked for FACS and a few commercial kits but they are out of my budget.
You can isolate small dense B cells using 'old school' method described by Phil Hodgkin and Marilyn Kehry which requires access to some hybridoma supernatants and guinea pig complement. Steps are (1) lyse RBCs, (2) deplete adherent cells (macrophages etc) by incubation on a plastic culture dish, (3) deplete T cells by complement-mediated lysis using CD4, CD8 and Thy1 hybridoma supernatant and (4) percoll density gradient separation to recover small dense B cells. Purity is >90%. Full method can be found in this reference:
Hodgkin, P.D., and M.R. Kehry. 1996. Methods for polyclonal B lymphocyte activation to proliferation and Ig secretion in vitro. Weir's Handbook of Experimental Immunology. L.A. Herzenberg, D.M. Weir, and C. Blackwell, editors. Blackwell Science, Cambridge. 89.1–13.
Good luck.
Sorry but I don't uderstand: do you whant to separate lymphocytes from blood? So do you still have the blood from spleen?
If you want to a pure population of B-cells, you will need to do some sort of antibody separation (which is somewhat expensive). If a crude isolation of lymphocytes will suffice, I would recommend performing a Ficoll gradient on the blood and spleen after a RBC lysis step. This will obtain all the mononuclear cells, predominantly lymphocytes, from these tissues. The spleen is roughly 80% B-cells, so if it is B-cells you are interested in that would be your best source. Just remember, there will still be T-cells and some other contaminating cells in your prep if you do not separate via specific B-cell markers.
Good luck,
~Adam
This is the reason why I have asked for :) Infact I use Ficoll gradient too, and you obtain also monocytes and other cells, so you must use a markers. On the other hand there are specific colums that contain a filter that is able to separate b-cells from others, with high performance, but are more expensive....Be sure that the centrifuge is the correct one: swinging bucket rotor is the best one and you must use it without blocking!
The first questions are what B cells populations are you after (or does it matter) and does ii matter if they are activated? That said, I don't know of any other state-of-the-art methods besides antibody separations (e.g. MACS or the like) or FACS. There are various old-school multi-step methods combining T cells depletion by rosetting and anti-CD3 (OKT3) antibody, removal of monocytes/macrophageds by adherance to plastic/glass wool, sephadex G10 etc. - look in methods of older papers for details.
You could try to make your own Ab-mediated magnetic separations for positive selection with anti-CD19 Ab plus relatively cheap commercially available magnetic beads. You must realize that you will loose CD19- B cells and are likely to activate those you do isolate. Using other markers like CD20 or CD22 will be less B cell specific than CD19, but still will enrich for B cells. CD20 may not "activate" the cells, though papers say anti-CD20 does cause Ca2+ mobilization, so do your due diligence. CD22 will not activate the cells. Use of CD40 as the B cell marker for enrichment obviously highly activates the cells.
To enrich B cells we used to adhere monocytes to plastic dishes, take the supernatant enriched for T and B cells and run that over a column filled with Nylon wool, which I think wako still sells (www.wako-chem.co.jp). T cells stick to that and you end up with a selection of enriched B cells. Not sure of the quality though...
You can isolate small dense B cells using 'old school' method described by Phil Hodgkin and Marilyn Kehry which requires access to some hybridoma supernatants and guinea pig complement. Steps are (1) lyse RBCs, (2) deplete adherent cells (macrophages etc) by incubation on a plastic culture dish, (3) deplete T cells by complement-mediated lysis using CD4, CD8 and Thy1 hybridoma supernatant and (4) percoll density gradient separation to recover small dense B cells. Purity is >90%. Full method can be found in this reference:
Hodgkin, P.D., and M.R. Kehry. 1996. Methods for polyclonal B lymphocyte activation to proliferation and Ig secretion in vitro. Weir's Handbook of Experimental Immunology. L.A. Herzenberg, D.M. Weir, and C. Blackwell, editors. Blackwell Science, Cambridge. 89.1–13.
Good luck.
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