Staining of cytokines in lymphocytes by modified and traditional methods

Objective To stain the cytokines in lymphocytes by modified and traditional methods,and compare the effectiveness by flow cytometry.Methods Peripheral blood samples of healthy volunteers were collected,from which PBMCs were isolated and subjected to intracellular staining by traditional and modified methods respectively.By the traditional method,the cells were stimulated,and stained on surface,then fixed and stained after the membranes were broken.However,by the modified method,the cells were directly fixed after stimulation,of which the membranes were broken,and the intramembrane and membrane surface staining were carried out simultaneously at various time points.The percentages of lymphocytes in the samples were determined by flow cytometry after the antibodies were labeled.Results The percentage of Th1 cells stained by modified method showed no significant difference with that by traditional method(P 0.05).However,the percentage of Th17 cells stained by modified method was significantly higher than that by traditional method(P 0.05),while the percentages of Th2 cells 0,48 and 96 h after the membranes were broken showed no significant difference(P 0.05).Conclusion By the modified method,the procedure for test was simplified.The modified method was beneficial to the quality control of flow cytometry,which might be used for staining of cytokines in lymphocytes instead of traditional method.