一种用于细胞数量定量的荧光测定方法及其在细胞黏附试验中的应用。

A fluorometric approach to the quantitation of cell number with application to a cell adhesion assay.

作者信息

Lewinsohn D M, Nickoloff B J, Butcher E C

机构信息

Department of Pathology, Stanford University School of Medicine, CA 94305.

出版信息

J Immunol Methods. 1988 May 25;110(1):93-100. doi: 10.1016/0022-1759(88)90087-7.

DOI:10.1016/0022-1759(88)90087-7

PMID:3131438

Abstract

We describe a fluorometric approach to the determination of cell number. Cells are covalently labeled with fluorescein isothiocyanate (FITC) under physiologic conditions. Cells are lysed with detergent, and released fluorochrome is assessed quantitatively with a fluorescence spectrophotometer. Fluorescence varies linearly with regard to cell number over a wide range of concentrations, and allows detection of as few as 8 X 10(3) cells/ml. We describe the effect of different time and temperature incubations of FITC-labeled cells on fluorescence intensity, and we use the method to analyze the binding of peripheral blood mononuclear leukocytes to interferon-gamma-treated keratinocytes. Finally, we demonstrate that the results in an adhesion assay are comparable to those obtained with 51Cr-labeled cells. Quantitative fluorescence analysis of cell number offers a safe, inexpensive, rapid, and accurate method of determining cell number without the biological hazard and waste disposal problems associated with radioactive labeling.

摘要

我们描述了一种用于测定细胞数量的荧光测定方法。在生理条件下，细胞用异硫氰酸荧光素（FITC）进行共价标记。用去污剂裂解细胞，并用荧光分光光度计对释放出的荧光染料进行定量评估。在很宽的浓度范围内，荧光与细胞数量呈线性变化，并且能够检测低至8×10³个细胞/毫升。我们描述了FITC标记细胞在不同时间和温度孵育对荧光强度的影响，并且我们使用该方法分析外周血单个核白细胞与经干扰素-γ处理的角质形成细胞的结合。最后，我们证明在黏附试验中的结果与用⁵¹Cr标记细胞所获得的结果相当。细胞数量的定量荧光分析提供了一种安全、廉价、快速且准确的测定细胞数量的方法，而不存在与放射性标记相关的生物危害和废物处理问题。