流式细胞术细胞周期分析小鼠肺组织样本制备方法的优化研究

Optimizing Study on Lung Tissue Sample Preparation of the Mouse and Cell Cycle Analysis by Flow Cytometer

目的 寻找细胞周期分析更好的样本处理方法.方法 以小鼠肺组织作为标本,采用四种方法（剪碎法、研磨法、剪碎结合酶消化法、研磨结合酶消化法）制备单细胞悬液,胎盘蓝染色检测细胞的存活率,经不同时间固定后的样本利用流式细胞仪进行细胞周期分析.结果 剪碎法结合酶消化法制备的单细胞悬液效果最佳;单细胞固定后两周内细胞各个时期所占比例基本相同.结论 小鼠肺组织单细胞制备采用剪碎法联合酶消化法优于其他三种方法,样本经固定处理后可以进行流式批量检测.

Objective To explore the mechanical methods for making lung tissue into single cell suspension. Methods Lung tissue samples preparation of the mouse got by four methods （＂cut＂ with sissors,grinding,＂cut＂ with sissors combined with enzymatic digestion,grinding with enzymatic digestion） were detected with microscrop and flow cytometry. The viability and morphologic changes of the single cell were observed by typan blue exclusion. Cell cycle of single cell suspension fixed at different time was analysed by FCM. Results The method of ＂cut＂ with sissors combined with enzymatic digestion was the best. The sample for cell cycle analysis by FCM could save for 2 weeks,and there were no significant difference in the per- centage of G0/G1% ,S% and G2/M. Conclusion The method of ＂cut＂ with sissors combined with enzymatic digestion for making lung tissue into single cell suspension was the best. Samples fixed can be analysed in volume-testing by the FCM.