L15 leibovitz s

Cells were seeded 3 days before the experiment and treated with 1µM myriocin following the procedure described in Jiménez-Rojo et al. and using 35 mm glass-bottom dishes obtained from MatTek (P35G-0.170 14-C). For staining of cells with the ER-flipper to visualize membrane tension, cells were incubated for 20 mins with 1µM ER flipper at 37ºC, washed afterwards with live imaging medium (Gibco L-15 Leibovitz's) and imaged in the same medium with 10% FBS. Fluorescence lifetime imaging microscopy (FLIM) was performed on a Leica SP8 DIVE, at 20 MHz, with lexc = 488 nm (white light laser) and 63x
oil immersion objective lens. To measure in 'inverted mode', around 200 μL of medium were kept on each dish and a cover slip was placed on top of the surface, then the dish was flipped upside down to image upright without drying out the cells. For analysis, a threshold is applied to exclude background pixels and a biexponential reconvolution model was used for the fitting. ROIs were manually selected from the images, outlining the ER membrane based on the signal of the probe and excluding the plasma membrane which was faintly stained and that could be easily excluded due to higher lifetime values.
Each point represents the average lifetime of different ROIs selected from 1 cell.