SEPARATION OF MOUSE BONE MARROW CELLS USING WHEAT GERM AGGLUTININ AFFINITY CHROMATOGRAPHY

Abstract

Mouse bone marrow cells were fractionated on columns of wheat germ agglutinin-Sepharose 6MB (WGA-Sepharose) and conditions established for specific binding and cell enrichment in the eluted fractions. A small proportion (7%) of the cells applied did not bind to the column and 60% of these were lymphocyte-like cells. Twice as many cells were eluted from the WGA-Sepharose column using N-acetyl-D-glucosamine (GlcNAc), and 80% of these cells were polymorphonuclear granulocytes and metamyelocytes. These cells were only released from the matrix in the presence of GlcNAc at high buffer flow rates (> 4 ml/min). Approximately 3 10⁸ bone marrow cells bound to 1 ml of WGA-Sepharose. At least 5 min were required for 90% binding of the cells and elution of the cells with GlcNAc required nearly 20 min at 24°. At 37° the elution of cells with CIcNAc was much faster (< 5 min) and a much larger percentage of cells (40-50%) was specifically eluted. Sodium azide (002%. w/v) did not prevent cells binding to WGA-Sepharose or alter the distribution of cells eiuted by GlcNAc, but did slightly increase cell yields. Elution of cells with different concentrations of GlcNAc gave fractions enriched in different cell types. Analysis of cell fractions with a fluorescence-activated cell sorter showed that the lymphocyte subpopulation which failed to bind to WGA-Sepharose had been depleted of cells with a high density of immunoglobulin on their surface. The lymphocytes with a high density of surface immunoglobulin were recovered in the cells released from WGA-Sepharose using mechanical agitation.