Anti cd9

Minimum essential medium α (MEMα), Opti-MEM alpha, and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). Total exosome isolation reagent, and lipofectamine RNAiMAX was purchased from Invitrogen (Foster city, CA, USA) and 5-(N,N-Dimethyl) amiloride hydrochloride (DMA), insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEXA), anti-TRPML1, and oil red O dye were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against PPARγ was purchased from Santa-Cruz Biotechnology, Inc. (CA, USA). Antibodies against C/EBPα, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). Exosome-depleted FBS and exosome marker antibodies including anti-CD9, anti-CD81, anti-CD63, and anti-Hsp70 were procured from System Biosciences (Palo Alto, CA, USA). Anti-LAMP1 antibody was purchased from Abcam (Cambridge, MA, USA).

Proteins were extracted from cells and purified exosomes with RIPA buffer containing 25 mM Tris HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (Thermo scientific, Rockford, lL USA). Protein concentration was measured with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins (30 μg) were loaded with 4%–12% gradient polyacrylamide gel (Invitrogen, Carlsbad, CA, USA) and then blotted onto PVDF membranes (Millipore, Watford, UK). Primary antibodies were incubated overnight at 4°C on the membrane and included: anti-CD9, anti-CD63, and anti-Hsp70 (1:1000 dilution; System Bioscience, Mountain View, CA, USA). The secondary antibodies were incubated for 1 h at room temperature and included: anti-rabbit (1:2,000 dilution, System Bioscience, Mountain View, CA, USA) for CD9, CD63, and Hsp70. Immunoreactive bands were imaged via luminescence using an IVIS imaging system (XenogenCorp., Alameda, CA, USA) and detection reagents (Roche, Nutley, NJ, USA).

Fresh extracellular vesicles were isolated from serum-free media conditioned by B16F0 cells for 24 hours and imaged with scanning electron microscopy (SEM) as described previously [18 (link), 21 (link)]. Briefly, EVs were isolated from cell-conditioned media by differential centrifugation as follows: 300xg for 10 minutes to remove cells, 2,600xg for 10 minutes to remove residual cells and debris, 10,000xg for 60 minutes to remove microvesicles, and 100,000xg for 2 hours to collect nano-scaled vesicles in pellets. The resulting pellet was resuspended, washed once in DPBS, and re-pelleted at 100,000xg for 2 hours. Once isolated, nano-scaled vesicles were resuspended in DPBS and kept on ice. EVs were then imaged using SEM. The abundance of proteins contained in B16F0 EVs were compared against B16F0 whole cell lysate by Western blot analysis using methods described previously [20 (link)]. Specifically, membranes were probed with rabbit anti-CD9, CD63, CD81, and Hsp70 antibodies (System Biosciences, Mountain View, CA), and mouse anti-β-actin and β-tubulin antibodies (Santa Cruz Biotechnology, Dallas, Texas). Proteins were transferred to Bio Trace PVDF membrane (PALL Life Sciences, Pensacola, FL) and detected using Pierce ECL Western Blotting Substrate (Life Technologies).