Exo green

Purified exosomes from either human or mouse plasma samples were labeled with green fluorescent linker PKH67 (Sigma-Aldrich, St. Louis, MO), Exo-Red, and Exo-Green (System Biosciences, Mountain View, CA), and further incubated at 37°C for 10 min, until the reaction was stopped by adding ExoQuick-TC reagent. Labeled exosomes were added to either differentiated human adipocytes (# PT-5006, Lonza, Walkersville, MD) or murine 3T3-L1 cells (ATCC, Manassas, VA) for 4 hours in a cell culture incubator at 37°C. Labeled exosomes were monitored for delivery into target cells using a Leica SP5 Tandem Scanner Spectral 2-photon confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL) with a 63× oil-immersion lens.

To evaluate fatty acid accumulation in renal tubular cells, BODIPY 493/503 from Life Technologies was performed as described previously [24 ]. Briefly, NRK-52E cells were treated with BSA, PA (250 μM), or OA (250 μM) for 24 h. The cells were washed with PBS, fixed in 4% paraformaldehyde, and stained with BODIPY 493/503. Stained NRK-52E cells were observed and imaged by a Leica confocal microscope.
To visualize the uptake of EVs into recipient cells, isolated EVs were resuspended in PBS. Approximately, 1.5 × 10⁸ EVs from HK-2 cells were fluorescently labeled with Exo-Green from System Biosciences (San Juan Capistrano, CA) and EVs from NRK-52E were labeled with PKH26 (Sigma-Aldrich). HK-2 or NRK-52E recipient cells were cultured on a coverslip in a 6-well plate until they reached 70% confluency. Confluent cells were washed twice with serum-free medium, and then exposed to 100 μl of labeled EVs resuspended in serum-free medium for 16 h. Cells were fixed with 4% paraformaldehyde, stained with DAPI and imaged by a Leica confocal microscope.