Isolation and characterization of cell cycle-enriched subpopulations of a murine macrophage cell line by centrifugal elutriation

Abstract

The cell cycle of the P388D1 murine macrophage line was delineated and suspensions of exponentially growing cells were separated by centrifugal elutriation into subpopuiations enriched in the various phases of the cycle. Analysis of both growth and labelled mitoses curves disclosed that the doubling and cell-cycle times were essentially identical (18.4 and 18.3 h), indicating that all cells were in cycle. In addition, G1+ 1 2 M was 4.3 h, whereas S phase and G2+ 1 2 M lasted about 12 and 1.5 h. The most homogeneous subpopulations of phase-enriched cells obtained by elutriation were cells in G1 and S, where purities (estimated by both labelling indices and analyses of DNA histograms obtained by flow cytometry) exceeded 80%. Isolation of G2+M-phase cells was not as efficient, although the purity of these subpopulations was consistently greater than of 50%, an approx. 10-fold enrichment over unseparated suspensions of cells. Comparison of IgG2a-Fc-receptor-mediated phagocytic activities among the phase-enriched subpopulations showed that cells in G2 had appreciably enhanced activity.