Membrane Tension Evaluation by FLIM

Membrane tension was evaluated by FLIM of polarized CFTR-CTL and CFTR-KD cultures incubated for 1 h at the basal side with the FliptR fluorescent probe (1 μl/ml, SC020; Spirochrome) under controlled temperature and atmosphere (37°C, 5% CO₂). FLIM imaging was performed using a Nikon Eclipse Ti A1R microscope equipped with a Time-Correlated Single-Photon Counting module from PicoQuant, as previously described (Colom et al, 2018 (link)), and a water immersion Apo LWD 40X/1.15 N.A. objective (Nikon). During acquisitions, cells were maintained at 37°C and 5% CO₂ with a micro-incubator (Okolab). FliptR fluorescence lifetime was determined for at least 10 positions, always at the same height within each Transwell filter. SymPhoTime 64 software (PicoQuant) was used to fit fluorescence decay data (from full images or regions of interest) to a dual exponential model after deconvolution for the instrument response function (measured using the backscattered emission light of a 1 μM fluorescein solution with 4M KI). Data were expressed as the mean ± SD of the mean.