Flow and ICAM-1 clustering synergistically increase endothelial membrane tension. (A-B) Fluorescence lifetime τ₁ images of FliptR in TNFα-activated HUVECs kept under static conditions or in the presence of low flow (1.2 dynes/cm²) or exposed to anti–ICAM-1 antibody beads or to anti–ICAM-1 crosslinking antibodies (ICAM-1 XL) without or together with low flow. The color bar corresponds to lifetime in nanoseconds. Scale bar, 15 µm. Corresponding lifetime mean values indicating membrane tension are shown in the bar diagram (B; n = 40 measurements from 5 independent experiments). (C-D) Representative pseudocolored Förster resonance energy transfer (FRET) images of MSS-expressing cells kept under static conditions or in the presence of low flow (1.2 dynes/cm²) or exposed to anti–ICAM-1 crosslinking antibodies (ICAM-1 XL) without or together with low flow (C). The color bar indicates YPet/ECFP emission ratio. Corresponding normalized YPet/ECFP emission ratio of Förster resonance energy transfer (FRET) biosensors indicating membrane tension are shown in the bar diagram (D; n = 15-22 measurements from 3 independent experiments). Shown are mean values ± SEM. n.s., nonsignificant; **P ≤ .01; ***P ≤ .001 (1-way ANOVA).