【摘要】 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。更多还原

【Abstract】 Objective: To optimize the cell pretreatment methods for detecting P-gp of MDR cells by flow cytometry(FCM) and increasing the detection rate of P-gp.Methods: By using JSB-1 with intramembranous antigen determinant as antibody,the positive rate as optimization index and MCF-7/A as model cells,cell pretreatment methods,such as the presence or Absence of FACSTM permeabilizing solution,the concentration of FACSTM permeabilizing solution,the time treating cells with FACSTM permeabilizing solution,the time incubating cells with JSB-1 working fluid,the time incubating cells with goat-anti-mouse IgG-FITC(GAMIF) solution,and the conditions for keeping JSB-1 working fluid and cell numbers,were optimized.Results: These cell pretreatment methods influenced on the positive rate of MCF-7/A to some extent.The relative optimization methods were as follows: cells numbered from 5×105 to 20×105 were incubated with the six-times-diluted solution of FACSTM permeabilizing solution for 10 min,and then incubated with JSB-1 working fluid for 75 min and GAMIF for 30 min,respectively,at 37℃;in addition,JSB-1 working fluid was conserved under 4 ℃ or was prepared freshly.Conclusion: By optimizing the cell pretreatment methods,the detection rate of P-gp of MDR cells could be increased significantly.And thus,the expression level of P-gp and the resistance degree of MDR cells could be revealed objectively by FCM.更多还原