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目的探讨化学染料羧基荧光素二乙酸盐琥珀酰亚胺酯(CFDA-SE)标记肿瘤细胞的最佳条件及其在细胞毒检测中的价值。方法用不同浓度的CFDA-SE标记K562(人红白血病细胞)、YAC-1(小鼠淋巴瘤细胞)、A375(人乳腺癌细胞)、MCF-7(人黑色素瘤细胞)不同肿瘤细胞系,并检测其0~24h的荧光强度,观察荧光强度变化的时间动力学,选择CFDA-SE标记细胞的最佳浓度;CFDA-SE标记细胞后孵育时间为1~6h,再用DNA染料碘化丙啶(PI)标记,流式细胞仪分析CFDA-SE和PI双标记细胞,并计算细胞死亡率,研究CFDA-SE对细胞的毒性。CFDA-SE标记时间分别为5、6、7、8、10、15min,测定标记不同时间的荧光强度和细胞的死亡率,选择最佳标记时间;用CFSE在最佳条件下标记靶细胞进行细胞毒检测实验。结果对于不同肿瘤细胞系,CFDA-SE标记的最佳浓度不同;CFDA-SE对细胞没有毒性,死亡率低于5%;最佳标记时间为8min。人外周血单个核细胞和BALB/c鼠脾淋巴细胞对肿瘤细胞K562、YAC-1均表现出杀伤活性,杀伤百分率随效靶比和共孵育时间的增加而增加。最佳效靶比为50∶1~25∶1,共孵育时间为2~4h。结论CFDA-SE具有标记细胞稳定,能用于细胞培养时间较长的研究,不影响细胞功能,适用于流式细胞仪检测细胞毒实验的优点,是一种很好的标记细胞的荧光素染料。
Objective To investigate the optimal conditions for the labeling of tumor cells by the chemical dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and its value in the detection of cytotoxicity. Methods Different tumor cell lines such as K562 (human erythroleukemia cells), YAC-1 (mouse lymphoma cells), A375 (human breast cancer cells) and MCF-7 (human melanoma cells) were labeled with different concentrations of CFDA- The fluorescence intensity of the cells was measured at 0-24 h. The kinetics of fluorescence intensity change was observed. The best concentration of CFDA-SE labeled cells was selected. After CFDA-SE labeling, the incubation time was 1 ~ 6 h, (PI), CFDA-SE and PI double-labeled cells were analyzed by flow cytometry, and the cell death rate was calculated to study the cytotoxicity of CFDA-SE. The CFDA-SE labeling time was 5, 6, 7, 8, 10 and 15 min, respectively. The fluorescence intensity and cell death rate at different time points were measured and the optimal labeling time was selected. The target cells were labeled with CFSE under optimal conditions Toxicity test. Results The optimal concentrations of CFDA-SE for different tumor cell lines were different. CFDA-SE was not toxic to cells and the mortality rate was less than 5%. The optimal labeling time was 8 min. Human peripheral blood mononuclear cells and splenic lymphocytes of BALB / c mice showed cytotoxicity against tumor cells K562 and YAC-1, and the percentage of killing increased with the increase of target ratio and incubation time. The best effective target ratio of 50: 1 ~ 25: 1, a total incubation time of 2 ~ 4h. Conclusion CFDA-SE has the characteristics of stable labeled cells, long time of cell culture, no effect on cell function, suitable for the detection of cytotoxicity by flow cytometry, and is a good fluorescent marker of cell labeling .