Measuring Adipocyte Membrane Tension in Mice

Primary visceral adipocytes from mice fed with standard diet or HFD for 7 days were isolated and incubated with 1 μM of the membrane tension probe Flipper-TR® (Tebu-bio, #SC020) for 30 min at 37 °C as described^{29 (link)}. The adipocytes were then washed 3 times with HBSS and imaged with a Leica-SP8 FLIM microscope. Excitation was performed using a pulsed 488 nm laser (Laser kit WLL2+pulse picker, Leica Microsystems) operating at 80 MHz, and the emission signal was collected from 549 to 651 nm with acousto-optical beam splitter (AOBS) using a gated hybrid (HyD SMD) detectors and a TimeHarp 300 TCSPC Module and Picosecond Event Timer (PicoQuant). SymPhoTime 64 software (PicoQuant) was then used to fit fluorescence decay data. To extract lifetime information, the photon histograms from membrane regions were fitted with a double exponential, and 2 fluorescence emission decay times (τ1 and τ2) are extracted. The longest lifetime with the higher fit amplitude τ1 is used to report membrane tension^{29 (link)}.