ABSTRACT:ObjectiveTo investigate factors that may affect the detection of intracellular cytokines with flow cytometry.MethodsSplenocytes from mice were disaggregated, and regulatory T cells (Treg) were detected by staining of CD4, CD25, and intranuclear transcription factor FOXP3.For the detection of TNFα, splenocytes were stimulated with PMA/Ionomycin, antiCD3/CD28 or PMA/Ionomycin plus antiCD3/CD28, respectively.Following activation, the cells were stained with antiCD3 APC and antiCD4 FITC, then intracellularly stained to detect TNFα after fixation and permeabilization.Controls were designed for both experiments.ResultsThe improper operations in the grinding, lysing erythrocytes, fixing and perming the cells all leaded to indistinct target cells of interest in the FSC/SSC dot plots, which affected the subsequent analysis.There were no significant differences in the percentages of lymphocytes, CD3+T lymphocytes, and TNFα producing cells between the fresh and frozen samples(P＞0.05).Expression of TNFα in cells stimulated by PMA/Ionomycin, antiCD3/CD28, or combined stimulation groups were significantly higher than controls(P＜0.05).ConclusionMany factors may affect the detection of intracellular cytokines by flow cytometry，including the spleen, lysing erythrocytes in the preparation of splenocytes suspension, fixing and perming the cells during dyeing, adjusting the compensation values, designing controlled trials and so on.Therefore, attentions should be paid to explore and select the suitable conditions for studying intracellular factors before testing.