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目的建立双光源流式细胞仪三参数细胞周期分析方法,同时检测两种细胞周期素和细胞DNA含量,分析非时相性的细胞周期素的表达。方法运用免疫荧光标记技术对两种cyclin标记,一种用直接法(FITC标记),另一种用间接法(RPE-Cy5标记的二抗),同时进行细胞核染色(荧光素DAPI),双光源二点激发,三波段定量检测,数据运用cellquest软件分析。mAMSA和mimosine诱导非时相性的细胞周期素表达。结果两种cyclin可同时检测,互不干扰,而且不需要荧光补偿,与单标相比无明显差别,cyclin及:DNA均可半定量分析。Cyclin A/ cyclin E/DNA分析可将对数生长期的MOLT-4细胞分为六个亚细胞群(G10、1le、G1l、S、G2、M),mimosine处理的细胞可见G1期cyclin B1表达,mAMSA处理的细胞有G2期cyclin E的异位表达。结论运用免疫荧光技术在流式细胞仪上三参数细胞周期分析可以快速定量检测两种cyclin和DNA含量,更精确地对细胞增殖进行分析。
OBJECTIVE: To establish a three-parameter cell cycle assay for dual-light source flow cytometry, and to simultaneously detect the contents of two kinds of cyclin and cellular DNA, and to analyze the expression of non-phase cyclin. Methods Two types of cyclin were labeled by immunofluorescence technique, one was direct method (FITC) and the other was indirect method (RPE-Cy5-labeled secondary antibody). Nuclei staining (fluorescein DAPI) Two-point excitation, three-band quantitative detection, data analysis using cellquest software. mAMSA and mimosine induce a non-temporal phase expression of cyclin. Results The two kinds of cyclin can be detected at the same time without interfering with each other and without the need of fluorescence compensation. There is no significant difference between the two kinds of cyclin and the single standard. Cyclin and DNA can be semi-quantitatively analyzed. Cyclin A / cyclin E / DNA analysis could divide MOLT-4 cells in logarithmic phase into six subcellular groups (G10, 1le, G1l, S, G2 and M) , mAMSA-treated cells had cyclin E G2 ectopic expression. Conclusions Three-parameter cell cycle analysis using flow cytometry using immunofluorescence assay can rapidly and quantitatively detect the contents of two kinds of cyclin and DNA and analyze the cell proliferation more accurately.