Hcx 63

Differentiated iBACs were stained with the fluorescent dye Lysotracker DeepRed (excitation/emission 647/668 nm) according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, adipocytes were incubated for 30 min at 37 °C with 50 nM Lysotracker. Afterwards, cells were harvested and stained with Ghost Dye Red 780 (Tonbo Biosciences, San Diego, CA, USA) to exclude dead cells and subsequently washed three times with PBS detached with trypsin, centrifuged at 1200 rpm for 5 min, and resuspended in PBS. To quantify the number of lysosomes, 1 × 10⁵ cells, gated for living cells, were analyzed using a Guava Easy Cyte 8 flow cytometer (Merck, Darmstadt, Germany). For confocal microscopy, cells were cultivated on cover slides in 6-well plates (Sarstedt, Nümbrecht, Germany) and stained with Lysotracker (described above). Afterwards, cells were imaged by confocal fluorescence microscopy using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica HCX 63 × 1.25 NA water immersion objective.

Cells (day 5 of differentiation) and mRFP-GFP-LC3 (ptfLC3) plasmid (# 21074, Addgene plasmid Cambridge, MA, USA) were used for electroporation (EP) with the Neon^® Transfection System (Thermo Fisher Scientific, Waltham, MA, USA) following the general protocol guidelines with the following modifications: iBACs were tryptic digested with 2.5% trypsin/0.5 U/ml collagenase D (Merck, Darmstadt, Germany) for 3.5 min and then resuspended in pre-warmed growth medium. Cells were pelleted at 300 ×g for 3 min and washed with PBS. Cells were counted and 450,000 cells and 2 μg plasmid DNA were mixed and used per 100 μl EP-tip. The EP was performed with the following program: 1400 V, 20 ms, 2 pulse and cells were plated on cover slides in 6-well plates in DMEM high glucose medium supplemented with FBS and without any antibiotics. The medium was changed to growth medium 24 h after EP and the cells (day 7 of differentiation) were subjected to confocal microscopy using a Leica HCX 63 × 1.25 NA water immersion objective on a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).