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。
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 rain 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. Condusions 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 .
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2006年第10期935-938,共4页
Chinese Journal of Laboratory Medicine
基金
吉林省科技厅重点资助项目(20050402-3
20040401-5)
吉林省科技厅国际合作资助项目(20040707-2)
2004年长春市科技局计划资助项目(2004218)
