Use of Size Fractionation of in Vitro‐Activated Human B Lymphocytes for Studies of Cell Cycle‐Dependent Growth Regulation

Cell cycle progression of in vitro-stimulated human B lymphocytes occurs a synchronously. In order lo allow detailed studies of growth Control G1. B cells Were stimulated with anti-μ and low molecular weight B-cell growth factor (LMW BCGF) for 50 h and subsequently separated into nine fractions of cells by means of centrifugal elutriation. As judged by volume profiles, activation antigen expression and DNA content, the cells in fractions t 4 were in early Io mid-G1, while fractions 5 7 mainly contained cells in late G1. and fractions 8 9 contained cells mainly in Sand G2, Cells in fractions 5–7 had passed the commitment point, as demonstrated by a high spontaneous incorporation of [3H]thymidine when recultured in medium alone. Moreover S-phase entry of these cells was largely unaffected by exogenous growth-promoting or growth-inhibitory signals. Cells in early (fractions 1 2) and intermediate fractions (fractions 3 4) showed a negligible spontaneous 3[H]thymidine incorporation, but a significant proportion of these cells progressed to S phase upon restimulation. Moreover, while IL-4 or the anti-CD40 MoAb G28- 5 potently stimulated cells in early and intermediate fractions, the responsiveness to LMW BCGF alone was obtained Just prior to the commitment point.