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目的利用二氯荧光黄二乙酸酯(2’,7’-Dichlorodihydrofluorescein diacetate,DCFH-DA)探针和4’,6’-二脒基-2-苯基吲哚(4’,6’-diamidino-2-phenylindole,DAPI)探讨影响活性氧(reactive oxidative species,ROS)和细胞核共染的因素,以优化最佳的研究方案。方法以RAW 264.7细胞株为研究对象,首先采用不同浓度的多聚甲醛分别固定10、30 min,1、2、4 h,进行ROS和DAPI检测;再用不同的固定试剂分别在ROS检测前和后固定,检测ROS荧光强弱;将DAPI在DCFH-DA探针加入前12、4、2、1 h,20 min加入和两者同时加入,检测DAPI染色情况;最后,DAPI在ROS检测前12 h加入,以不加DAPI组为对照,分别用LPS和H_2O_2刺激产生ROS并检测,计算其荧光数和平均荧光强度,利用SPSS18.0软件进行t检验分析。结果不同固定浓度、固定试剂、固定顺序的结果均表明,固定虽然会增强DAPI的染色,但同时会减弱ROS的荧光强度;活细胞DAPI染色时,随着染色时间的延长,核着色强度和均匀度都更好;DAPI染色12 h后检测ROS,对ROS的荧光数和平均荧光强度都没有统计学影响(P>0.05)。结论 DCFH-DA探针法检测ROS荧光时不能与细胞固定连用;DAPI在DCFH-DA探针孵育前12 h加入,并不影响ROS荧光的产生和检测,该研究方案可有效实现ROS与DAPI的共染。
OBJECTIVE: To investigate the effect of 4 ’, 6’-diamidino-2-phenylindole using 2’7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) 2-phenylindole, DAPI) to explore the factors affecting reactive oxidative species (ROS) and nuclear co-staining, in order to optimize the optimal research program. Methods The RAW 264.7 cell line was used as the research object. ROS and DAPI were detected by using different concentrations of paraformaldehyde for 10, 30, 1, 2 and 4 h, respectively. After fixation with DAPI, the fluorescence intensity of ROS was detected. DAPI was added at 12,4,2,1 h and 20 min before addition of DCFH-DA probe, and DAPI staining was detected. h to join, with no DAPI group as a control, were stimulated with LPS and H_2O_2 produce ROS and detected, calculate the fluorescence number and average fluorescence intensity, using SPSS18.0 software for t test analysis. Results The results of different fixed concentrations, fixed reagents and fixed sequences showed that although the fixation enhanced the staining of DAPI, the fluorescent intensity of ROS decreased at the same time. When DAPI staining of living cells, with the extension of dyeing time, the nuclear staining intensity and uniformity The detection of ROS at 12 h after DAPI staining had no statistical significance on the fluorescence and average fluorescence intensity of ROS (P> 0.05). Conclusions DCFH-DA probe method can not detect the fluorescence of ROS and can not be used with cells fixedly. DAPI can be added 12 h before DCFH-DA probe incubation, which does not affect the production and detection of ROS fluorescence. This protocol can effectively achieve ROS and DAPI Total dye.