Analyzing Membrane Dynamics in Hepatocytes

Membrane dynamics was analyzed as described (Golfetto et al., 2013 (link)). Briefly, primary hepatocytes from Lpcat3^fl/fl and Lpcat3^fl/fl; Albumin-Cre mice were isolated as described (Rong et al., 2013 (link)). Cells were incubated with 1.8 mM Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene; Invitrogen) at 37°C for 30 min. Cells were rinsed with phosphate-buffered saline (PBS), and fresh culture medium was added. Spectral data were acquired with a Zeiss LSM710 META laser scanning microscope coupled to a 2-photon Ti:Sapphire laser (Mai Tai, Spectra Physics, Newport Beach, CA) producing 80-fs pulses at a repetition of 80 MHz with two different filters: 460/80 nm for the blue channel and 540/50 nm for the green channel. Spectral data were processed by the SimFCS software (Laboratory for Fluorescence Dynamics). The GP value was calculated for each pixel using the two Laurdan intensity images (460/80 nm and 540/50 nm). The GP value of each pixel was used to generate the pseudocolored GP image. GP distributions were obtained from the histograms of the GP images.