Density gradient electrophoresis of mouse spleen lymphocytes: separation of T and B cell fractions

Preparative electrophoresis in an isotonic Ficoll--sucrose density gradient has been employed for the separation of mouse (C57Bl/6J) spleen lymphocyte subpopulations. The separated cells were pooled into six fractions according to their relative position (Rp) within the total cell distribution. In general, the high mobility cells were identified as T lymphocytes. These cells exhibited immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated mouse anti-theta globulin and responded in vitro to phytohemagglutinin stimulation. The low mobility cells were activated in vitro by E. coli lipopolysaccharide and showed immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated anti-mouse Ig which is typical of mouse B lymphocytes. Both T and B cells were completely isolated from each other in certain fractions of very high and very low mobility, respectively. Overlapping of the two distributions was observed in the intermediate mobility fractions. The method which utilizes an inexpensive commercially available apparatus should be useful for the preparation of other lymphocyte subpopulations differing in surface charge.