Hcs lipidtox red

A431 cells, seeded onto glass-bottom dishes (Nunc LabTek 4-well chambered coverglass), were first labeled with 50 μg/ml Alexa Fluor 647-dextran (10,000 MW; Thermo Scientific) supplemented with 200 μM oleic acid/BSA in 5% LPDS to label late endosomal organelles and to induce LDs. The cells were then pulse-labeled for 2 h with 50 μg/ml BC LN-LDL in serum-free DMEM. During the last 30 min of LDL labeling, HCS LipidTox Red or HCS LipidTox Deep Red (1:1000; Thermo Scientific) was added to the medium to label LDs. The cells were then washed and chased in serum-free CO₂-independent medium (Gibco) for the indicated times and imaged by live-cell confocal microscopy. Imaging was performed on a Leica TCS SP8 X attached to a motorized DMi8 inverted microscope with ×63 HC PL APO CS2 water objective (1.20 NA). Experiments were performed at 37 °C in CO₂-independent medium (Gibco) supplemented with HCS LipidTox Red/Deep Red (1:1000) in a fully enclosed temperature-controlled environmental chamber. Data were acquired with Leica LAS X (Leica Microsystems). The fraction of BC residing in dextran-positive LEs and LipidTox-positive LDs was quantified from background-subtracted images with ImageJ by using Mander’s overlap coefficient as a measure of colocalization.

HepG2 cells were cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, catalog no. c11995500BT) supplemented with 10% fetal bovine serum (FBS, Gibco, catalog no. 16000-044) and 1% penicillin-streptomycin (HyClone, catalog no. 30010). A GFP1-10 sequence was inserted into the N terminal of human PLIN2. The resulting GFP1-10-Plin2 was then inserted into a pCDH-CMV-MCS-EF1-Puro vector. The N terminal of human TOM20 (1-33 aa) was used as a mitochondria targeting sequence. Mito-GFP11 was inserted into a pCDH-CMV-MCS-EF1-Puro vector. Lentivirus was packaged as previously described^{53 (link)}. Wildtype HepG2 cells were infected by lentiviruses to stably express mito-GFP11 and GFP1-10-Plin2. The LD–mitochondria contact reporter cells were selected and maintained in DMEM supplemented with 2 μg ml⁻¹ puromycin. Then, the cells were grown on lacey 200 mesh gold EM grids (T10012Au, Beijing XXBR Technology Co., Ltd) overnight to promote adherence and spreading. Mitochondria were stained with 1 μM MitoTracker Deep Red (M22426, Thermo Fisher Scientific) at 37 °C for 30 min. For the validation of the LD–mitochondria contact reporter, LDs were stained with HCS LipidTox red (H34476, Thermo Fisher Scientific) at a dilution of 1:1,000 at 37 °C for 30 min.