【摘要】 目的:探讨不同实验条件对流式细胞术细胞周期分析结果的影响。方法:采用大鼠C6细胞系,检测在不同的乙醇浓度、固定方式、染色时间、固定时间、样品流速及细胞浓度等实验条件下对细胞周期分析结果的影响。结果:75%乙醇固定细胞效果最佳,直接用无水乙醇固定会引起细胞不同程度的黏连聚集,浓度过低则导致固定不充分,从而影响分析结果(P<0.05);将细胞沉淀用磷酸缓冲盐溶液(phosphate buffered saline, PBS)重新悬浮后加入乙醇固定效果明显(P<0.05);乙醇固定24 h以上效果佳,长时间-20℃保存对结果无影响(P>0.05);细胞浓度过高可因染色不充分而影响分析结果(P<0.05);样品流速加大对细胞周期分析结果的影响差异有统计学意义(P<0.05);样品在染色后1～12 h内细胞周期的结果差异无统计学意义(P>0.05),<30 min可因染料未充分结合而影响分析结果(P<0.05)。结论:在仪器设备状态良好的前提下,推荐先将细胞用PBS重悬,然后加入3倍体积的预冷无水乙醇,轻轻混匀打散细胞后置-20℃固定24 h以上,按每1×106个细胞加0.5 mL染液室温避光染色1 h以上以低流速上样检测为佳。更多还原

【Abstract】 Objective: To investigate the effect of different experimental conditions on cell cycle analysis by flow cytometry.Methods: C6 rat cell line was used to detect the effects of ethanol concentration, fixation method, staining time, fixation time,sample flow rate and cell concentration on cell cycle analysis results. Results: 75% ethanol fixed cells had the best effect,direct use of absolute ethanol fixed cells will cause different degrees of adhesion and aggregation, too low concentration will lead to inadequate fixation, thus affecting the analysis results(P<0.05); the cell precipitation was resuspended with phosphate buffered saline(PBS) and then fixed with ethanol(P<0.05); the effect of ethanol fixation for more than 24 hours was good, and long-term storage at-20 ℃ had no effect on the results(P>0.05). The results were affected by insufficient staining when the cell concentration was too high(P<0.05); the effects of increased sample flow rate on the results of cell cycle analysis had siginificant difference(P<0.05); the results of cell cycle analysis were not significantly different between 1 h and 12 h after staining(P>0.05), and the results could be affected by insufficient dye binding when the concentration was less than 30 min(P<0.05). Conclusion: On the premise that the equipment is in good condition, it is recommended to use PBS to resuspend the cells, then add 3 times volume of precooled absolute ethanol, gently mix and disperse the cells, and then fix them at-20 ℃for more than 24 hours. Add 0.5 mL dye solution to each 1 ×106 cells and stain the cells in dark at room temperature for1 hour. It is better to use low flow rate for sample detection.更多还原