Fig 1: Flotillin expression is required for conjugate formation and T cell signaling. a Representative flow cytometry dot plots of wildtype and Flotillin1/2 KO Jurkat T cells conjugated to SEE pulsed Raji B cells and stained with cell viability dye and CFDA, respectively. b Percentage of stable T–B cell conjugates determined as violet dye and CFDA double positives, relative to total cell population. c Flow cytometry quantification of WT and flotillin1/2 KO Jurkat T cells activated with soluble CD3ε and CD28 and fixed prior or 5, 10, and 20 min after activation. Cells were stained with antibodies against phosphorylated (left) TCRζ-pY142, (middle) Zap70-pY493, and (right) ERK-pY202/204. d Representative immunofluorescence microscopy images of WT and flotillin1/2 KO Jurkat T cells stained with antibodies against nuclear factor of activated T-cells (NFAT) and e the integrated NFAT fluorescence intensity over the Hoechst-stained nucleus in WT and flotillin1/2 KO Jurkat T cells activated or not with anti-CD3ε and CD28 antibodies. f Representative immunofluorescence microscopy images of WT and flotillin1/2 KO Jurkat T cells stained with antibodies against T-cell-specific adaptor protein (TSAd) and g the integrated TSAd fluorescence intensity over the Hoechst-stained nucleus in WT and flotillin1/2 KO Jurkat T cells activated or not with anti-CD3ε and CD28 antibodies. Scale bars, 5 µm. Data obtained from three or more independent experiments. Error bars indicate mean (±SEM). *p < 0.01; **p < 0.001; ****p < 0.00001; Mann–Whitney t-test