Dfc360

3T3-L1 pre-adipocytes were seeded at a density of 5 × 10³ cells per well in 96-well plates. Cellular H₂S levels were quantified using live cell imaging as described [8 (link), 21 , 22 (link)]. After differentiation and treatments, cells were incubated with 10 µM of the H₂S sensitive probe AzMC in HBSS buffer and further incubated at 37 °C for 1 h. The specific fluorescence of the dye was visualized using a Leica DFC360. FX microscope and images were captured with Leica Application Suite X (LAS X) software (Leica Biosystems Nussloch GmbH, Germany). Images were analyzed with ImageJ software (v. 1.8.0; NIH, Bethesda, Maryland, USA) and data and graphed with GraphPad Prism 8 (GraphPad Software Inc.; San Diego, California, USA).

Fluorescent images were captured with a Leica AF6000 epifluorescence microscope connected to a DFC360 camera while bright-field images were captured using a Leica DLMB microscope coupled to a DFC480 camera. Exposure settings were kept constant throughout to enable comparisons between samples. Figures were edited using Adobe Illustrator and Photoshop CS6.

KM cells were incubated for 24 h with serum-free or EV-depleted FBS prior fixation with 4% PFA for 15 min. Cells were washed three times with PBS, before formaldehyde quenching with NH4Cl for 10 min at RT. Cells were permeabilized with 0.3% PBS-Triton X for 15 min followed by blocking with fish block Triton X 0.3% for 1 h at RT. Cell were incubated with primary antibodies overnight at 4 °C (Arl13b, 1:800, #17711-1-AP, Proteintech; GT335, 1:200, #AG-20B-0020-C100, Adipogen Life Sciences; Cyclin D1, 1:200, #55506, Cell Signaling). Cells were washed three times with PBS before incubation with appropriate fluorescent conjugated secondary antibodies (anti-mouse IgG Alexa Four 555, 1:400, Thermo Fisher Scientific; anti-rabbit IgG Alexa Fluor 488, 1:400, Thermo Fisher Scientific), counterstained with DAPI (1:8000, #6843, Carl Roth) and TRITC-Phalloidin (1:400, Thermo Fisher Scientific). The cell was imaged on a Leica CTR6000 confocal microscope, with a DM6000 B laser, the monochrome digital camera DFC360 FX and Leica image Software BlindDeblur Algorithm, one iteration step. All images were processed in Fiji ImageJ 64 v5 software.