Objective: To establish a method for detecting intracellular IL-2 in human peripheral blood γδΤ cells.
Methods: Healthy Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated, then stimulated with low molecular peptide antigen of Mycobacterium tuberculosis (Mtb-Ag), and a high proportion of γδΤ cells was obtained. PBMCs and γδΤ cells were excited with phorbol myristate acetate(PMA) and incomycin (IM). Protein transportion inhibitors (Brefeldin A, BFA) were used to block the secretion of cytokines and saponins 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.
Results: Compared with those with 5 min and 25 min of membrane permeabilization, CD3+ T cells with 15 min has the highest expression of CD69 (87.82 ± 2.28%) (F=112.805, P<0.001). Compared with γδΤ group, γδΤ+PMA+IM group and PBMC+PMA+IM+BFA group, γδΤ cells with the stimulant of PMA, IM and blocker of BFA (γδΤ+PMA+IM+BFA) had the highest expression of intracellular IL-2 (50.65 ± 6.25%) (F=321.071, P<0.001).
Conclusion: Flow cytometry is an appropriate method for the detection of intracellular IL-2 in γδΤ cells, and it may be applicable to detect other cytokines in γδΤ cells.Author(s): Keqiang Wang, Yanqiang Hou, Yanchao Duan, Yiren Wang, Xiangqi Li