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目的:探讨喉癌细胞株Hep-2中侧群细胞分选的条件优化。方法:取对数生长期的Hep-2细胞,予胰酶消化后,制成1×106个/ml单细胞悬液。①实验组中加入Hoechst33342至终浓度分别为5、9、10、11μg/ml,37℃水浴90min。②取单细胞悬液,分别于37°水浴中孵育Hoechst 50、70、90、110、130min。③实验组喉癌细胞悬液加入Hoechst后,分两管分别孵育于37°水浴及37°恒温箱中。两管保持Hoechst浓度及细胞密度一致。④取汇合度分别为100%、70%的2瓶细胞,同上制成单细胞悬液。染色结束后,重悬PBS中,上流式细胞仪进行检测鉴定。以上各组均设阴性对照组于Hoechst染色前加维拉帕米,37℃中孵育30min,其他各条件保持与相应实验组一致。结果:①当Hoechst33342浓度分别为5、9、10、11μg/ml时,SP比例分别为(39.96±0.24)%、(26.23±0.39)%、(18.79±0.02)%、(19.01±0.14)%;死细胞比例分别为(30.45±0.63)%、(49.90±0.42)%、(50.12±0.68)%、(64.16±0.39)%。②Hoechst在37°水浴中孵育90min时,流式细胞图形最典型,SP比例为(17.37±1.42)%;③孵育于37°水浴较孵育恒温箱中,实验组未被Hoechst33342标记的细胞分别为(18.67±0.45)%、(22.6±0.50)%;④汇合度90%以上较汇合度70%的细胞,SP比例由(19.08±0.46)%降到(16.073±0.52)%。以上结果均经SPSS 13.0分析,差异有统计学意义(P<0.05)。结论:在流式细胞仪分选喉癌Hep-2侧群细胞中Hoechst33342的最佳浓度为10μg/ml,最佳孵育时间为90min,且水浴优于恒温箱中孵育,细胞密度最好选择80%~90%。
Objective: To investigate the condition optimization of side population cell sorting in Hep-2 cell line. Methods: Hep-2 cells in logarithmic growth phase were digested with trypsin and then made into 1 × 106 cells / ml single cell suspension. ① Hoechst33342 was added to the experimental group to the final concentration of 5, 9, 10, 11μg / ml, 37 ℃ water bath for 90min. ② take a single cell suspension, respectively, in a 37 ° water bath incubation Hoechst 50,70,90,110,130 min. ③ Experimental group laryngeal cancer cell suspension added Hoechst, were divided into two tubes were incubated in 37 ° water bath and 37 ° incubator. Two tubes maintained the same Hoechst concentration and cell density. ④ Take the confluence of 100%, respectively, 70% of the 2 bottles of cells, ibid made a single cell suspension. After staining, resuspended in PBS, the detection by flow cytometry. The above groups were set negative control group before Hoechst staining plus verapamil, incubated at 37 ℃ for 30min, the other conditions remain consistent with the corresponding experimental group. Results: ① When the concentration of Hoechst33342 was 5, 9, 10 and 11μg / ml, the SP ratios were (39.96 ± 0.24)%, (26.23 ± 0.39)%, (18.79 ± 0.02)% and (19.01 ± 0.14) (30.45 ± 0.63)%, (49.90 ± 0.42)%, (50.12 ± 0.68)% and (64.16 ± 0.39)%, respectively. Hoechst incubated in 37 ° water bath for 90min, the most typical flow cytometry, SP ratio was (17.37 ± 1.42)%; ③ incubated in 37 ° water bath incubation incubator, the experimental group were not Hoechst33342 labeled cells were ( 18.67 ± 0.45)% and (22.6 ± 0.50)%, respectively. (4) The percentage of SP decreased from (19.08 ± 0.46)% to (16.073 ± 0.52)% in more than 90% confluence. The above results were analyzed by SPSS 13.0, the difference was statistically significant (P <0.05). CONCLUSION: The optimal concentration of Hoechst33342 in side population cells of Hep-2 in laryngeal squamous cell carcinoma by flow cytometry is 10μg / ml, the optimal incubation time is 90min. % ~ 90%.