SEPARATION BY COUNTERFLOW CENTRIFUGAL ELUTRIATION AND ANALYSIS OF T-LYMPHOCYTIC AND B-LYMPHOCYTIC CELL-LINES IN PROGRESSIVE STAGES OF CELL-DIVISION CYCLE

The method of counterflow centrifugal elutriation (CCE) facilitates th e non-invasive separation of proliferating cells into the progressive stages of the cell division cycle. We present here detailed protocols for the separation of primary lymphocytes and lymphocytic cell lines i ncluding Jurkat, a mature human T-cell line, Ramos, a human B-cell lin e, WEHI-231, a murine B-cell lymphoma, and stimulated human peripheral T-cells into progressive stages of the cell division cycle by counter flow centrifugal elutriation. Protocols for using the elutriator to co ncentrate large volumes of cells prior to separation, the preparation of highly enriched lymphocyte populations at progressive stages throug h the cell division cycle and conversion parameters from low to high v olume rotors are described. Simple dual-staining methods of BrdUrd inc orporation and propidium iodide staining for DNA content and subsequen t flow cytometry are detailed. Together with [H-3]thymidine incorporat ion data these provide a very accurate determination of cell cycle pos ition of the separated populations.