Validation of two assays for determination of neutrophil oxidative burst and neutrophil phagocytosis

来源 :中国药理学与毒理学杂志 | 被引量 : 0次 | 上传用户:nature_shcn
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OBJECTIVCE To investigate the altered oxidative burst activity and phagocytic activity,respectively.METHODS Both PHAGOBURST assay,which determines the percentage of neutrophils that undergo an oxidative burst,and PHAGOTEST assay which determines the percentage of neutrophils that engulf fluorescein-labelled opsonized E.coli bacteria,were performed following the kits’ procedures with a flow cytometer.Heparinized blood samples from cynomolgus monkeys were analyzed for intra-assay and intra-animal precision,sample stability and inter-subject variability.RESULTS Intra-assay coefficients of variation(CV) for the phagoburst assay were 0.4 %(1 mol·L-1,one male animal) and 0.2%(1F,one female animal),while intra-assay CVs for phagocytosis assay were 3.4 %(1 mol·L-1) and 0.7%(1F).Intra-animal CVs over 3 d for the phagoburst assay were 8.7%(1mol·L-1) and 4.1%(1F),whereas intra-animal CVs for the phagocytosis test were 1.2%(1mol·L-1) and 5.2%(1F).Inter-subject variability was determined with 4 male animals and 4 female animals.Besides,the heparinized blood samples were stable for both oxidative burst activity and phagocytic activity after kept at room temperature for 8 h and for 24 h,respectively.CONCLUSION All the parameters tested throughout the study fulfilled the criteria of acceptance.Both phagoburst assay and phagocytosis assay are methodologically acceptable for the use in the analysis of phagocytic function in cynomolgus monkeys. OBJECTIVCE To investigate the altered oxidative burst activity and phagocytic activity, respectively. METHODS Both PHAGOBURST assay, which determines the percentage of neutrophils that undergo an oxidative burst, and PHAGOTEST assay that determines the percentage of neutrophils that engulf fluorescein-labeled opsonized E. coli bacteria , were performed following the kits’ procedures with a flow cytometer. Heparinized blood samples from cynomolgus monkeys were analyzed for intra-assay and intra-animal precision, sample stability and inter-subject variability .RESULTS Intra-assay coefficients of variation (CV) for The phagoburst assay was 0.4% (1 mol·L-1, one male animal) and 0.2% (1F, one female animal), while intra-assay CVs for phagocytosis assay were 3.4% % (1F) .Intra-animal CVs over 3 d for the phagoburst assay were 8.7% (1 mol·L-1) and 4.1% (1F), and the intra- animal CVs for the phagocytosis test were 1.2% (1 mol·L- 1) and 5.2% (1F) .Inter-subject variability was determined with 4 male animals and 4 female animals. Besides, the heparinized blood samples were stable for both oxidative burst activity and phagocytic activity after kept at room temperature for 8 h and for 24 h, respectively. CONCLUSION All the parameters tested throughout the study fulfilled the criteria of acceptance.Both phagoburst assay and phagocytosis assay are methodologically acceptable for the use in the analysis of phagocytic function in cynomolgus monkeys.
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