QUANTITATIVE CYTOCHEMICAL ASPECTS OF A COMBINED FEULGEN-NAPHTHOL YELLOW S STAINING PROCEDURE FOR SIMULTANEOUS DETERMINATION OF NUCLEAR AND CYTOPLASMIC PROTEINS AND DNA IN MAMMALIAN-CELLS
QUANTITATIVE CYTOCHEMICAL ASPECTS OF A COMBINED FEULGEN-NAPHTHOL YELLOW S STAINING PROCEDURE FOR SIMULTANEOUS DETERMINATION OF NUCLEAR AND CYTOPLASMIC PROTEINS AND DNA IN MAMMALIAN-CELLS
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DOI：
10.1016/0014-4827(75)90386-9
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发表时间：
1975-01-01
影响因子：
3.7
通讯作者：
ZETTERBERG, A
中科院分区：
文献类型：
--
作者：
GAUB, J;AUER, G;ZETTERBERG, A
采用feulgen -萘酚黄S （NYS）联合染色法同时测定细胞核和细胞质蛋白和DNA。按照这个程序，在NYS染色之前进行Feulgen染色。主要研究结果如下：单独进行NYS染色后，发现与细胞结合的NYS量与细胞干质量密切相关。乙醇丙酮固定细胞的相关系数为0.99，甲醛固定细胞的相关系数为0.95。Feulgen染色过程没有显著改变这种密切相关性，在乙醇丙酮中为0.92，在甲醛固定细胞中为0.94。然而，固定方法和Feulgen水解类型会影响每单位干质量结合的NYS的绝对数量。在Feulgen过程中，细胞损失物质，特别是在酸水解阶段。最适合Feulgen程序的水解类型（5 N HCl, 22°C, 60 min）导致乙醇-丙酮固定细胞中相当大的干质量损失。这种损失在甲醛固定的细胞中较小（15%），并且与水解前细胞的干质量密切相关（相关系数0.99）。在甲醛固定细胞中，Feulgen法后的干质量是测定固定细胞真实干质量的好方法。这进一步证明了NYS与Feulgen染色细胞的结合与Feulgen处理前这些细胞的干质量密切相关（相关系数0.95）。3.3。在标准化条件下（5 N HCl, 22°C, 60 min，甲醛固定和酸水解）使用Feulgen-NYS联合染色程序时，发现在具有不同增殖活性的各种类型的哺乳动物细胞中，每单位干重有一定量的NYS结合到细胞核和细胞质蛋白上。随后的NYS染色未发现Feulgen DNA测定受到定量影响。从本研究的结果来看，在标准化的条件下，Feulgen-NYS联合染色法可以作为一种可靠的定量方法来测定哺乳动物细胞的细胞核和细胞质蛋白质和DNA。
The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained:1.1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis.2.2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95).3.3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity.4.4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining.From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.