A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle  

双光源流式细胞术三参数细胞周期分析(英文)

作　　者：冯永东[1] 陶德定[1] 覃吉超[1] 高纯[1] 申漫里[1] 冷艳 余源[1] 龚建平[1] 

机构地区：[1]武汉华中科技大学同济医学院附属同济医院肿瘤研究所/分子医学中心,430030

出　　处：《The Chinese-German Journal of Clinical Oncology》2005年第2期76-82,共7页中德临床肿瘤学杂志（英文版）

基　　金：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) grants from the Science Foundation of Ministry of Public Health, China (No. 202-01-06).

摘　　要：To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.

关 键 词：cell cycle flow cytometry CYCLIN three-parameter ANALYSIS 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]