Isolation of haemopoietic spleen colony forming cells

A simplified and more widely applicable modification of the recently developed methodology for sorting and collecting purified populations of the pluripotent haemopoietic spleen colony-forming cell, CFU-S, is described. Based on their relatively low density and high affinity for wheat germ agglutinin, CFU-S are collected using a fluorescence activated cell sorter. Normal bone marrow cells are labelled with flurorescein labelled wheat germ agglutinin (WGA - FITC) and then subjected to a density cut on metrizamide. Cells with density less than 1.080 gm. ml-1 are sorted on a FACS-IV instrument. Highly fluorescent cells with medium forward and low perpendicular light scatters are collected. These cells are then re-sorted to the same criteria as for the first sort. This double sorting procedure gives a population of cells which, corrected for spleen seeding efficiency, contains about 90% CFC-S. Mixed CFC, in vitro, are also enriched but from the ratios of mixed to spleen CFC, and of 11 to 8 day spleen colony forming ability, there is a clear selection for the earlier, more primitive and/or non-cycling Go-phase CFC-S. This is confirmed by direct observation of autoradiographic labelling indices.