Sorting of B lymphoblasts based upon cell diameter provides cell populations enriched in different stages of cell cycle.

Here we report analysis and correlation of changes in cell size and cycle state resulting from exposure of murine B lymphocytes to the mitogens lipopolysaccharide (LPS) and dextran sulfate (DxSO4). Cell cycle changes are assessed by flow cytofluorometric analysis of acridine orange stained cells. Cell diameters are determined by flow cytometric analysis of the pulse-width (time of flight) of the axial light extinction signal. Results indicate that within 12 h of exposure of B cell populations to these mitogens, cells displaying increased diameter and containing increased RNA can be detected. Under these conditions, increased RNA content is considered indicative of G0 to G1 transition or entry into cell cycle (Darzynkiewicz et al., 1976). Progressive increases in cell size and transition through G1, S, G2, and M occur in parallel during 48 h of culture with mitogens. Sorting of cells based upon size followed by cell cycle analysis reveals a direct correlation between cell size and cycle phase. Specifically, cells 4.5-5.5 microns in diameter are in primarily G0. Cells 5.5-7.0 microns in diameter are in early G1. Populations of cells 7.0-10 microns in diameter are comprised of late G1 and S phase cells. Populations of cells 10-12 microns in diameter consist of S, G2, and M phase cells. The importance of this correlation is discussed in view of needs to more rigorously define B cell populations for investigations of biochemical events of and accessory cell requirements for activation of B lymphocytes.