A method for evaluating phagocytosis of mouse peritoneal macrophages by flow cytometry / 中国免疫学杂志

ABSTRACT

To explore a sensitive , stable and handleable method for evaluating phagocytosis of mouse peritoneal macrophages by flow cytometry , and get a set of optimized solutions.Methods: The peritoneal macrophages obtained from ICR mice were divided into two part.One part was used directly ,and another part was 1∶1 diluted.Three fluorescent microsphere concentrations were used (5×106/well,1×107/well and 1.5×107/well).Incubation time were respective 1 h,1.5 h and 2 h.The adherent cells were digested by enzyme or cell scraper.The percentage of phagocytic cells ( PP) and the phagocytic index ( PI) were determined by flow cy-tometry.To verify and confirm the reliability of experiment conditions , effect of JKS on phagocytosis of mouse macrophages were evaluated with flow cytometric assays and chicken red blood-cell method.Results:The higher concentration of fluorescent microspheres meant PP and PI were higher.When cell concentration was 1×105-2×105 ml-1 ,incubation time was 1.5 h,concentration of fluorescent microspheres was 1.5 ×107/well,the PP and PI were the highest (89.87%,1.54).When incubation time was 2 h,the PP and PI declined(57.71%,1.51).Effect of cell concentration on the PP and PI were negatively correlated with fluorescent microspheres .After adherent macrophages were digested by trypsin+EDTA,the PP and PI were 44.51%,0.68.The PP and PI were 37.92%,0.57 after di-gestion by EDTA.The results were lower than using cell scraper.The PP(1 485 mg/kg group) of JKS were higher than control group that were evaluated with flow cytometric assays and chicken red blood-cell method.The difference was statistically significant ( P<0.05 ).Conclusion: These are the optimized solutions for the experiment such as the concentration of peritoneal macrophaes is (1-2)×105,the incubation time is 1 h and the concentration of fluorescent microspheres is 1×107/well.