流式细胞术同时检测BrdU与细胞表面抗原方法的改进  

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

作　　者：于鸿晶[1] 吴明媛[1] 向砥[1] 俞雁[2] 韩伟[1] 

机构地区：[1]上海交通大学药学院再生组学课题组,200240 [2]上海交通大学农业与生物学院上海市兽医生物技术重点实验室

出　　处：《免疫学杂志》2010年第9期813-815,共3页Immunological Journal

基　　金：上海市科学技术委员会(075407071;09540700600)

摘　　要：目的改善用流式细胞仪检测BrdU和细胞膜表面抗原同时标记的方法。方法小鼠腹腔注射BrdU100mg/kg,3h后取胸腺制成胸腺单细胞悬液。细胞经CD4、CD8表面抗体标记后,2%多聚甲醛溶液4℃固定过夜,0.1%Triton x-100/0.1%柠檬酸钠/PBS(pH7.2)冰上孵育2min,50U DNaseI37℃孵育30min,BrdU抗体标记,上机检测。结果此方法在细胞表面抗原与BrdU同时检测时,既能够有效的区分BrdU+、BrdU-细胞,同时BrdU抗原标记的处理过程不影响检测细胞表面抗原的检测。结论该方法可用于流式检测BrdU与表面抗原的同时标记,适用于多细胞亚群增殖的检测。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.

关 键 词：流式细胞术 细胞增殖 BRDU 表面标记 胸腺 

分 类 号：R392.11[医药卫生—免疫学]