IDENTIFICATION OF RESTING CELLS BY DUAL-PARAMETER FLOW-CYTOMETRY OF STATIN EXPRESSION AND DNA CONTENT

Statin, a 57-kDa nuclear protein, has been recognized as a unique mark er of quiescent (G(0)) cells; specific monoclonal antibodies (MoAb) ag ainst statin have been produced and used to label resting cells in tis sue sections and in cultured cells, We present an improved method for the identification of G(0) cells by dual-parameter flow cytometry of s tatin expression and DNA content, The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocy tochemical detection of statin; among them, 70% ethanol was selected b ecause this fixation procedure is suitable for DNA staining with inter calating dyes and is routinely used for the immunolabeling of prolifer ation markers (such as proliferating cell nuclear antigen [PCNA] and K i-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody ( S-44), and indirect fluorescein isothiocyanate immunolabeling, cells w ere counterstained for DNA. with propidium iodide and analyzed by dual -parameter flow cytometry, In cells from several animal sources (rat t hymocytes and Cb glioma cells, mouse 3T3 cells, and human MCF-7 cells) , under different experimental conditions, the expression of statin wa s found to correlate inversely with that of PCNA and Ki-67, and with t he BrdUrd labeling index, In dual-parameter now scattergrams, G(0) (st atin positive) cells can be discriminated from the potentially cycling (statin negative) G(1) cells, i,e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool b oth for monitoring changes in the resting cell fraction and for invest igating the process of G(0)-G(1) transition in unperturbed and drug-tr eated cell populations. (C) 1995 Wiley-Liss, Inc,