The tonsils are superior to the peripheral blood as sources of mononuclear cells for B-cell purification.
Various methods for B-cell purification from mononuclear cells of the peripheral blood and tonsils were evaluated. L-leucine methyl ester was found to be the best method for monocyte depletion when B cells were isolated. Purification of B cells by negative selection methods from peripheral blood mononuclear cells (PBMCs) resulted in cell populations containing less than 40% B cells. This was partly due to significant increase in the relative number of large granular lymphocytes during the purification procedures. However, when T cells and monocytes were depleted from tonsillar mononuclear cells (TMCs), 84.6% of the remaining cells were B cells. The cell populations isolated from PBMCs and TMCs by negative selection methods differed from each other also in their responsiveness to pokeweed mitogen. Positive selection methods, such as panning and cell sorting by flow cytometer, resulted in good purities with both PBMCs and TMCs. However, the cell yields with the positive selection methods were always very low. It is concluded that the tonsils should be favoured as sources of B cells when negative selection methods are used. When peripheral blood B cells are wanted, positive selection methods should be favoured.
Punnonen
J
J
Department of Medical Microbiology, Turku University, Finland.
Viljanen
M K
MK
Eerola
E
E
Eskola
J
J
D001402:B-Lymphocytes / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D002850:Chromatography, Gel; D005434:Flow Cytometry; D006801:Humans; D008212:Lymphocyte Depletion; D009000:Monocytes / Q000276:immunology; D014066:Palatine Tonsil / Q000276:immunology*; D012397:Rosette Formation; D013601:T-Lymphocytes / Q000276:immunology