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目的:优化流式细胞术(FCM)检测肿瘤耐药细胞中P-糖蛋白(P-gp)的细胞前处理方法,提高细胞中P-gp的检出率。方法:采用抗原决定簇在细胞膜内的单抗JSB-1,以阳性百分比为优化指标,耐阿霉素人乳腺癌细胞系MCF-7/A为模型细胞,对FACSTM透化溶液的有无、FACSTM透化溶液的浓度、FACSTM透化溶液处理细胞的时间、鼠抗JSB-1抗体的孵育时间、羊抗鼠IgG-FITC(GAMIF)抗体的孵育时间、JSB-1抗体工作液的保存条件、以及细胞数量进行优化。结果:上述细胞处理方法对MCF-7/A的阳性百分比均有不同程度的影响;相对最佳的细胞处理方法为:5×105-20×105个细胞用FACSTM透化溶液的6倍稀释液处理10min,37℃下与JSB-1抗体孵育75min、与GAMIF抗体孵育30min,JSB-1抗体工作液于4℃保存或新鲜配制。结论:对FCM检测前的细胞处理方法进行优化,能显著提高肿瘤耐药细胞中P-gp的检出率,因而更能真实、客观地反映肿瘤耐药细胞的P-gp表达水平及耐药程度。
Objective: To optimize flow cytometry (FCM) for the detection of P-glycoprotein (P-gp) in tumor-resistant cells and to improve the detection rate of P-gp in cells. Methods: The monoclonal antibody JSB-1 with antigenic determinant in the cell membrane was used as the optimization index, and the doxorubicin-resistant human breast cancer cell line MCF-7 / A was used as a model cell. The effects of FACSTM permeabilization solution, The concentration of FACSTM permeabilization solution, the time of FACSTM permeabilization solution treatment, the incubation time of mouse anti-JSB-1 antibody, the incubation time of goat anti-mouse IgG-FITC antibody, the storage condition of JSB-1 antibody working solution, As well as the number of cells to optimize. Results: The above-mentioned cell treatment methods had different degrees of positive effect on the percentage of MCF-7 / A positive cells. The best cell treatment method was: 5 × 105-20 × 105 cells with 6-fold dilution of FACSTM permeabilization solution Treated with JSB-1 antibody at 37 ℃ for 75 min and incubated with GAMIF antibody for 30 min. The working solution of JSB-1 antibody was stored at 4 ℃ or freshly prepared. Conclusion: The optimization of cell-processing methods before FCM detection can significantly increase the detection rate of P-gp in drug-resistant cells, which can reflect the P-gp expression level and drug resistance in tumor-resistant cells more objectively and objectively degree.