【摘要】 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。更多还原

【Abstract】 Objective To study the property and methodology of CFDA-SE on labeling tumor cells.Methods To select the optimal concentration, we assay the fluorescent intensity by labeling four tumor cell lines with CFDA-SE on different concentrations and with the incubation time of 0～24 h.To analyze the toxicity of CFDA-SE on cells which were incubated at different time points from 1 to 6 h, we perform subsequent DNA-labeling with PI and collect 1 000 target events to calculate the percentage of cell death.To select the optimal labeling time , we labeled cells with CFDA-SE for 5、6、7、8、10、15 min and determined the fluorescence intensity and cell death respectively.Results The optimal labeling concentration of CFDA-SE for each cell line was variable, CFDA-SE was non-toxic on cells since the proportion of cell death was below 5%, 8 min incubation with CFDA-SE was selected as the optimal labeling time.The cytotoxic activity showed a significant and positive correlation with E∶T ratio and incubation period.The optimal E∶T ratio and incubation period were 50∶1～25∶1 and 2～4 h.Conclusions The advantages of CFDA-SE are as follows:long-term incubation period is allowed. CFDA-SE fluorescence dye is not toxic and can be used by flow cytometry. CFDA-SE is found to be a suitable fluorochrome for labeling cells更多还原