Separation by counterflow centrifugal elutriation and analysis of T- and B-lymphocytic cell lines in progressive stages of cell division cycle

Abstract

The method of counterflow centrifugal elutriation (CCE) facilitates the 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 including Jurkat, a mature human T-cell line, Ramos, a human B-cell line, WEHI-231, a murine B-cell lymphoma, and stimulated human peripheral T-cells into progressive stages of the cell division cycle by counterflow centrifugal elutriation. Protocols for using the elutriator to concentrate large volumes of cells prior to separation, the preparation of highly enriched lymphocyte populations at progressive stages through the cell division cycle and conversion parameters from low to high volume rotors are described. Simple dual-staining methods of BrdUrd incorporation and propidium iodide staining for DNA content and subsequent flow cytometry are detailed. Together with [ 3H]thymidine incorporation data these provide a very accurate determination of cell cycle position of the separated populations.