N of EVs released by GEN2.two cells was performed using the labelling protocol created by Sargiacomo and colleagues [41]. This protocol was according to cell treatment using the commercially available BODIPY FL C16 fatty acid (4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid) (Life Technologies, Monza, Italy), hereafter indicated as Bodipy C16, a fluorescent lipid that labels the cells, ultimatelyViruses 2022, 14,5 ofproducing fluorescent vesicles. Briefly, the fluorescent lipid was resuspended in methanol at 1 mM final concentration and stored at -20 C in aliquots of 150 . Prior to use, each and every aliquot was dried beneath nitrogen gas at room temperature, resuspended with 30 of 20 mM KOH to avoid the formation of micelles and to promote its solubilisation, heated for ten min at 60 C and finally resuspended in 70 of PBS containing two of bovine serum albumin (BSA). For pulse-chase studies, 1 107 GEN2.two cells were metabolically labelled with Bodipy C16 at various occasions and concentrations, as reported inside the text. Importantly, to favour the uptake on the fluorescent probe, the treatments were performed using complete medium supplemented with only 0.3 FBS. Afterwards, cells have been MEK1 Inhibitor Source washed with 1PBS to remove lipid excess, and complete culture medium supplemented with 10 FBS was added. The fluorescence intensity of GEN2.2 cells was evaluated by flow cytometry evaluation and reported in terms of mean fluorescence intensity (MFI), and then observed by confocal microscopy. For the isolation and quantification of fluorescent EVs, 1 107 GEN2.2 cells had been seeded in 75 cm2 flasks and incubated for 2 h at 37 C, with three.five of Bodipy C16 in 5 mL of medium supplemented with 0.3 FBS. Then, cells had been washed with 1PBS and resuspended in 12 mL of complete culture medium supplemented with 10 FBS, containing or not myrNefSF2 w.t. The FBS added to the medium was previously SIRT2 Inhibitor manufacturer ultracentrifuged overnight for 18 h at one hundred,000g in a SW41 Ti rotor (Beckman Coulter, Brea, CA, USA), to remove the EVs ordinarily present in serum. two.5. Extracellular Vesicle Purification EVs had been isolated from identical volumes (12 mL) of cell conditioned and nonconditioned handle media, which had been harvested immediately after 20 h and processed following the already described solutions for EV purification [42]. Briefly, cell cultures or culture medium, utilized as a manage, were centrifuged at 290g for 7 min to remove cells and then at 2000g for 20 min to get rid of cell debris. Subsequently, supernatants underwent differential centrifugations consisting of a initial ultracentrifugation at 15,000g for 20 min to isolate large/medium EVs (hereafter known as microvesicles). To isolate tiny EVs (known as exosomes), supernatants have been then harvested and ultracentrifuged at one hundred,000g for three h. The pelleted vesicles were left for 20 min on ice and after that resuspended in 12 mL of 1PBS and ultracentrifuged once again at one hundred,000g for three h. All ultracentrifugation actions have been performed at four C applying a SW41 Ti rotor (Beckman Coulter, Brea, CA, USA). Isolated exosomes and microvesicles had been lastly resuspended in 10000 of PBS with protease and phosphatase inhibitors (1 mM sodium orthovanadate, 20 mM sodium fluoride, 1 /mL leupeptin and pepstatin A, two /mL aprotinin and 1 mM phenylmethylsulfonyl fluoride (PMSF)) and stored at 4 C till counting by flow cytometry and additional analyses. 2.6. Quantification of Vesicles by Flow Cytometry Flow cytometry of Bodipy-labelled EVs was performed on a Gallios.
