The clinical application of flow cytometry for detecting intracellular IL-2 in γδΤ cells
Wang Keqiang1,Hou Yanqiang2,Ran Zhangshen1，Li Xiangqi1，Duan Yanchao1
1Department of Clinical Laboratory,Affiliated Hospital of Taishan Medical University,Tai′an 271000,China；2Department of Clinical Laboratory，Songjiang District Central Hospital of Shanghai,Shanghai 201600,China
Abstract：ObjectiveTo establish a method for detecting intracellular IL-2 in human peripheral blood γδΤ cells.MethodsHealthy human peripheral blood mononuclear cells (PBMCs) were isolated,and then stimulated with low molecular peptide antigen of mycobacterium tuberculosis (Mtb-Ag).A high proportion of γδΤ cells were obtained.PBMCs and γδΤ cells were excited with phorbol myristate acetate (PMA) and ionomycin(IM).Protein transport inhibitor (Brefeldin A,BFA) was used to block the secretion of cytokines and saponin was taken for membrane permeabilization.Flow cytometry was applied to detect the expression of CD69 in CD3+ T cells to explore the appropriate time of membrane permeabilization.Then flow cytometry was implemented to detect the expression of intracellular IL-2 in γδΤ cells.Analysis of Variance （ANOVA） and Least Significant Difference（LSD） test were conducted to determine the statistical difference.ResultsCD3+ T cells with 15 minutes of membrane permeabilization had the highest expression of CD69 (87.82±2.28)% compared with those with 5 minutes and 25 minutes (F=112.81,P<0.05).γδΤ cells with the stimulation of PMA,IM and blocker of BFA (γδΤ+PMA+IM+BFA) had the highest expression of intracellular IL-2 (50.65±6.25)% compared with γδΤ group,γδΤ+PMA+IM group and PBMC+PMA+IM+BFA group（F=321.07,P<0.05）.ConclusionFlow cytometry is an appropriate method for the detection of intracellular IL-2 in γδΤ cells,and it may be applicable to detecting other cytokines in γδΤ cells.
