Kamalia N, McCulloch C A, Tenenbaum H C, Limeback H
Faculty of Dentistry, University of Toronto, Ontario.
J Histochem Cytochem. 1992 Jul;40(7):1059-65. doi: 10.1177/40.7.1376742.
Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.
在细胞化学中，细胞制剂通常在固定后用透射光进行分析，并与诸如偶氮偶联染料等试剂反应。对于用荧光细胞化学染料染色的细胞悬液，也可以通过流式细胞术对细胞进行分析和分选。我们利用细胞化学反应中产生的固红紫LB的强烈红色荧光，对大鼠骨髓基质细胞中的碱性磷酸酶（AP）表达进行流式细胞术分析。通过修改单细胞悬液的染色方案，我们证明，与用固红TR染色相比，该方法具有特异性，能够区分整个群体中不同水平的酶表达，并允许对异质性细胞群体进行酶动力学研究。该方法被应用于研究糖皮质激素地塞米松（Dx）对细胞增殖和AP表达的影响。在低AP表达细胞中，10(-8) M的Dx处理使[3H] - 胸腺嘧啶核苷标记指数从3.85%提高到5.24%（p < 0.01）。相反，高AP表达细胞未被[3H] - 胸腺嘧啶核苷标记。本文报道的染色和分析方法有助于检测、分离和定量表达碱性磷酸酶活性的骨髓基质细胞亚群。这些实验证明了流式细胞术作为传统细胞化学方法辅助手段的价值。
