Method improvement on the simultaneous determination of BrdU and membrane antigen by flow cytometry assay

This study is aimed to improve the method of simultaneous detection of BrdU and membrane antigen by flow cytometry assay. BrdU incorporation in mice was done by intraperitoneal injection of BrdU at a dose of 100 mg/kg with a interval of 1.5 hours. Three hours after the first injection,the thymi were isolated by dissection and the single-cell suspensions were prepared. After labeling with CD4 and CD8 markers,the cells were fixed with 2% paraformaldehyde/PBS at 4℃ overnight. The cells were then per-meabilized with 0.1% Triton x-100/ 0.1% Na-Citrate/PBS (pH7.2) on ice for 2 min,and treated with 50 units of DNaseI at 37 ℃ for 30 min. Then the cells were stained with anti-BrdU antibody. The results showed that the BrdU+ and BrdU-cells can be clearly separated by detecting BrdU fluorescence. Besides,the progress of marking BrdU has no effect on surface membrane antigen detection. We concluded that this method could be used for the simultaneous detection of BrdU and membrane antigen by flow cytometry assay,which may be well used to analyze the cells subgroup proliferation in varies field.