中德临床肿瘤学杂志（英文版）2005，Vol.4Issue(2)：76-82,7.
双光源流式细胞术三参数细胞周期分析
A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle
摘要
Abstract
Objective: To set up a three-parameter method for cell cycle analysis by two-laser flow cytometer, which can detect two types of cyclin plus DNA content in one measurement, and that analyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysis of two types of cyclins and DNA content simultaneously in individual cells by two-laser flow cytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. A Triton-X100 permeabilization procedure was optimized for detection of two types of cyclins. One cyclin was stained directly with a FITCconjugated monoclonal antibody (mAb), and the other, indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochrome DAPI. mAMSA and mimosine treated MOLT-4cells were used to test this three-parameter method. Results: Permeabilization with 0.5% Triton-X100 in PBS containing 1% BSA for 5 min on ice provided optimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It was found that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured with no compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growing MOLT-4 cells could be dividedinto 6 compartments (G1o, G1e, G1l,S, G2, and M) simultaneously, allowing for analysis of cell cycle phase specific perturbations without the necessity of cell synchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine and cyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleled expected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only be applied to measure unscheduled vs. expected cyclin expression but may also be used to estimate the fraction of cycling cells in up to 6 cell populations.关键词
细胞周期/细胞增殖/细胞周期蛋白/流式细胞术/三参数Key words
cell cycle/flow cytometry/cyclin/three-parameter/analysis引用本文复制引用
冯永东,陶德定,覃吉超,高纯,申漫里,冷艳,余源,龚建平..双光源流式细胞术三参数细胞周期分析[J].中德临床肿瘤学杂志（英文版）,2005,4(2):76-82,7.基金项目
This project was supported by grants from China Key Basic Research Program Grant (No. G1998051212), the National Natural Sciences Foundation of China (No. 39670265, 39730270 and 39725027) and grants from the Science Foundation of Ministry of Public Health （No. G1998051212）
