Pellicciari C, Mangiarotti R, Bottone M G, Danova M, Wang E
Dipartimento Biologia Animale, Centro di Studio per l'Istochimica del CNR, Pavia, Italy.
Cytometry. 1995 Dec 1;21(4):329-37. doi: 10.1002/cyto.990210404.
Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G0) 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 G0 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 dual-parameter flow scattergrams, G0 (statin positive) cells can be discriminated from the potentially cycling (statin negative) G1 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 G0-G1 transition in unperturbed and drug-treated cell populations.
他汀蛋白是一种57千道尔顿的核蛋白，已被公认为静止（G0）细胞的独特标志物；针对他汀蛋白的特异性单克隆抗体（MoAb）已被制备出来，并用于标记组织切片和培养细胞中的静止细胞。我们提出了一种通过对他汀蛋白表达和DNA含量进行双参数流式细胞术来鉴定G0细胞的改进方法。通过使用静止和增殖的人成纤维细胞作为模型细胞系统，建立了合适的技术条件。几种固定剂被证明适用于他汀蛋白的免疫细胞化学检测；其中，选择70%乙醇是因为这种固定方法适用于用嵌入染料进行DNA染色，并且常用于增殖标志物（如增殖细胞核抗原[PCNA]和Ki-67）以及溴脱氧尿苷（BrdUrd）掺入的免疫标记。在用去污剂使细胞通透、暴露于抗他汀蛋白抗体（S-44）并进行间接异硫氰酸荧光素免疫标记后，用碘化丙啶对细胞进行DNA复染，并通过双参数流式细胞术进行分析。在来自几种动物来源的细胞（大鼠胸腺细胞和C6胶质瘤细胞、小鼠3T3细胞和人MCF-7细胞）中，在不同的实验条件下，发现他汀蛋白的表达与PCNA和Ki-67的表达呈负相关，与BrdUrd标记指数也呈负相关。在双参数流式散射图中，可以将G0（他汀蛋白阳性）细胞与潜在增殖（他汀蛋白阴性）的G1细胞区分开来，即在具有相同DNA含量的细胞亚群中。这种方法可被视为一种强大的工具，既能监测静止细胞亚群的变化，又能研究未受干扰和经药物处理的细胞群体中G0-G1转变的过程。
