Identification of resting cells by dual‐parameter flow cytometry of statin expression and DNA content

Abstract

Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (Go) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of Go cells by dual-parameter flow cytometry of statin 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 immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dualparameter flow scattergrams, Go (statin positive) cells can be discriminated from the potentially cycling (statin negative) GI cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of Go+ transition in unperturbed and drug-treated cell populations. 0 1995 Wiley-Liss, Inc.

All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells

“…This protein is present in resting Go cells, and its expression declines when cells re-enter GI, early before the transition into S-phase. The immunolabelling with antistatin monoclonal antibodies is therefore a unique technique for identifying quiescent cells by light microscopy or by flow cytometry, as we recently demonstrated (Pellicciari et al, 1995).…”

“…The effect of 1I" M ATRA on cells treated for different times (24 h to 10 days) was assessed first by evaluating DNA content histograms and then by dual-parameter determination of either BrdU incorporation or immunopositivity for cell-cycle related proteins vs DNA content. As a marker for proliferating cells, the nuclear antigen Ki-67 was used (Gerdes et al, 1984), whereas the presence of statin was considered as an indication of acquired, kinetic quiescence (Wang, 1985;Wang and Krueger, 1985;Pellicciari et al, 1995). This approach enables the rapid estimation of the percentage of actually (or potentially) cycling cells and of quiescent cells (Pellicciari et al, 1995).…”

“…As a marker for proliferating cells, the nuclear antigen Ki-67 was used (Gerdes et al, 1984), whereas the presence of statin was considered as an indication of acquired, kinetic quiescence (Wang, 1985;Wang and Krueger, 1985;Pellicciari et al, 1995). This approach enables the rapid estimation of the percentage of actually (or potentially) cycling cells and of quiescent cells (Pellicciari et al, 1995). To detect apoptotic cells, we assessed the presence of the sub-G, peak in DNA histograms and the labelling of membrane phosphatidylserine (PS) residues by Annexin V. Pellicciari et al (1995Pellicciari et al ( , 1996.…”

“…This approach enables the rapid estimation of the percentage of actually (or potentially) cycling cells and of quiescent cells (Pellicciari et al, 1995). To detect apoptotic cells, we assessed the presence of the sub-G, peak in DNA histograms and the labelling of membrane phosphatidylserine (PS) residues by Annexin V. Pellicciari et al (1995Pellicciari et al ( , 1996. Anti-Ki-67 monoclonal antibodies were from Dako (Glostrup, Denmark), while monoclonal antistatin antibodies were a kind gift of Dr Eugenia Wang (Wang and Krueger, 1985).…”

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

“…The proliferative response of PBMC after CSFV in vitro re-stimulation was measured by bromodeoxyuridine incorporation [1,16]. We adapted this method for use in flow cytometry and double or triple labeling in order to evaluate the specific proliferation of B and T cell sub-populations.…”