Factors Influencing Flow Cytometry-based Cell Cycle Detection and Analysis of Various Immune Cell Subpopulations
10.13865/j.cnki.cjbmb.2024.09.1302
- VernacularTitle:流式细胞术检测细胞周期影响因素及不同免疫细胞亚群周期分析
- Author:
Dan LIU
1
;
Jie ZHANG
;
Zheng-Yang GUO
;
Li-Xiang XUE
;
Yu-Qing WANG
Author Information
1. 北京大学第三医院医学创新研究院基础医学研究中心,北京 100191
- Keywords:
flow cytometry(FCM);
cell cycle;
propidium iodide(PI);
immune cell subpopulations
- From:
Chinese Journal of Biochemistry and Molecular Biology
2024;40(9):1308-1316
- CountryChina
- Language:Chinese
-
Abstract:
Cell cycle analysis is essential for determining the cell proliferation state,studying cell func-tions,and evaluating drug effects.Flow cytometry is a commonly used method for cell cycle detection,with propidium iodide(PI)being the most widely used fluorescein.Nevertheless,various factors may af-fect the test results.Additionally,comparing distributions of immune cell subpopulations across different cell cycle stages can provide valuable insights into immunological responses and disease conditions.In this research,the B16-F10 cell line was used to study the impact of three factors on PI staining-based cell cycle detection:fixation settings,sample preparation conditions,and software analysis.To fix cells,it is suggested to suspend 3 × 106 cells in 300 μL of pre-cooled PBS,add 700 μL of 100％ethanol drop-wise,fix overnight at 4℃ or-20℃,and collect at a low flow rate(400-600 events/s)to ensure collec-tion of at least 3 000 singlets.Furthermore,dual-labeling with 5-ethynyl-2'-deoxyuridine(EdU)and PI can accurately distinguish cell cycle phases.And various immune cell subpopulations can be analyzed without cell sorting by combining surface marker staining with PI and Ki-67 staining.Here we review fac-tors affecting cell cycle identification using the PI staining method and provide a standard operating proto-col for the experiment.We established the method to combine EdU with PI for cell cycle detection and a-nalysis of immune cell subpopulations,thus expanding the approaches for cell cycle detection.
