SEPARATION OF HUMAN LYMPHOID CELLS INTO G1, S, AND G2 CELL CYCLE POPULATIONS BY USE OF A VELOCITY SEDIMENTATION TECHNIQUE

Abstract

Human lymphoid tissue culture cells can be separated according to cell size and corresponding cell cycle phase with a velocity sedimentation centrifugation method employing a continuous 5–20% wt/wt Ficoll gradient. A 7-fold increase in streaming limit was achieved by placing a buffer zone of isosmolar 5% Ficoll on top of the gradient before application of the cell load. The various pooled populations of cells from upper, middle, and lower areas of the gradient were characterized using autoradiographic, TCA-precipitable 3H]thymidine incorporation, and Fuelgen microspectrophotometric methods. The upper range of the gradient contains cells in the G1 cell cycle phase; the lower range, cells in the G2 phase; cells found in the middle of the gradient belong largely to the S phase of the cell cycle. These gradient-separated cell pools contained relatively little contamination with cells from other phases of the cell cycle and, when explanted from the gradient into fresh growth media, showed growth patterns characteristic of synchronized cell populations. This system of cell separation provides a useful tool for investigating the relationship of the cell cycle to surface membrane and metabolic characteristics in human lymphoid cell culture systems.