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目的探讨活体染料羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)标记兔眼虹膜色素上皮(IPE)细胞的可行性。方法使用酶消化加机械分离的方法分离培养兔眼IPE细胞,将传3~4代的IPE细胞用2.5、5、10、20、40μmol/L浓度的CFSE分别作用1、5、10min进行染色,通过观察染色后的细胞荧光强度和细胞贴壁率筛选出最理想的标记条件。采用流式细胞术追踪检测标记的IPE细胞的荧光强度变化。用免疫荧光抗体标记法检测此染色剂有无渗漏以及是否对周围细胞着染。结果浓度为20μmol/L的CFSE37℃下水浴作用1min为IPE细胞染色、观测、研究的最适条件。流式细胞仪和荧光显微镜观察结果显示,CFSE对IPE细胞染色可持续4周以上,未发生染料渗漏或转染其它细胞,且此浓度的CFSE不影响细胞的性状。结论活体染料CFSE是一种染色率高,追踪时间长,应用简便安全可靠的标记兔IPE细胞的新方法。
Objective To investigate the feasibility of biotinylated carboxyfluorescein acetoacetate succinimidyl ester (CFSE) labeled rabbit eye iris pigment epithelium (IPE) cells. Methods The rabbit eye IPE cells were isolated and cultured by enzymatic digestion and mechanical separation. IPE cells passaged from 3 to 4 passages were stained with CFSE at concentrations of 2.5, 5, 10, 20 and 40 μmol / L for 1, The optimal labeling conditions were screened by observing the stained cell fluorescence intensity and cell attachment rate. Fluorescence intensity changes of the labeled IPE cells were tracked by flow cytometry. Immunofluorescent antibody labeling was used to detect the presence or absence of bleedings and staining of surrounding cells. Results The optimum conditions of IPE staining, observation and research were as follows: the concentration of 20μmol / L CFSE in water bath at 37 ℃ for 1min. Flow cytometry and fluorescence microscopy showed that CFSE staining of IPE cells for more than 4 weeks, no dye leakage or transfection of other cells, and this concentration of CFSE does not affect cell traits. Conclusion The living dye CFSE is a new method of labeling rabbit IPE cells with high staining rate, long tracking time, simple and safe application.