[A methodology study on flow cytometric analysis of cell DNA stained with DAPI and Hoechst33342].
OBJECTIVE
To discuss a simple method for flow cytometric analysis of cell DNA stained with DAPI and Hoechst33342.


METHODS
HT 29 cells stained with DAPI, Hoechst33342 or PI were measured by BD FACSAria and the percentages of cells in G0/G1, S and G2/M phases with three staining methods, then the results were analyzed and compared. Before measurement we monitored the quality of DNA analysis of flow cytometer through UV beads QC experiment and analyzed the standard chicken erythrocyte nuclei (CEN) and calf thymocyte nuclei (CTN) stained with DAPI and Hoechst33342.


RESULTS
CV value of UV peak was 2.4 after QC experiments. There were 4 peaks on CEN histograms and the ratios of peak channel mean of G2/G1, G3/G1, and G4/G1 were about 2, 3, and 4 respectively. Both CV values of the first peak were 2.4. There were 2 peaks on CTN histograms and the ratio of peak channel mean of G2/G1 was 1.97, and CV value of G0/G1 2.4. The complete cell cycle of HT29 cells stained with DAPI, Hoechst33342 or PI was showed entirely, CV values were 3.40, 3.02 and 4.42, respectively, and the percentages of cells in G0/G1 were 60.86%, 60.22% and 60.81%,respectively, in S, 28.85%, 29.70% and 29.82%,respectively, and in G2/M, 10.29%, 9.09% and 9.37%, respectively. The results by the three methods showed no difference.


CONCLUSIONS
This method for measurement of cellular DNA content is a simple and efficient approach to determining cell cycle and can be the first choice when using flow cytometer with 355 nm UV.
Liu
Xi-juan
XJ
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Centralab, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing 100142, China. liuxj-2003@163.com
Ding
Hui-rong
HR
Zhang
Hong
H
D001562:Benzimidazoles; D002453:Cell Cycle; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D019073:HT29 Cells; D006801:Humans; D007211:Indoles
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride