Extracellular Vesicle Isolation from HEK-293T Cells

For isolating EVs, HEK-293T cells were conditioned in DMEM media supplemented with 10% EV-free FBS. FBS was depleted of EVs by ultrafiltration using Amicon Ultra-15 100 kDa filter devices (Merck Millipore, Darmstadt, Germany) as described previously [25 (link)]. The isolation of EVs from conditioned media was performed according to the protocol described by Heath et al. with modifications [26 (link)]. Conditioned media were consequently centrifuged at 300× g (10 min), 2000× g (10 min), and 10,000× g (10 min). Clarified media were applied onto columns filled with Macro-Prep DEAE Resin (Bio-Rad, Hercules, CA, USA), washed with 20 column volumes (CV) of buffer containing 50 mM of HEPES and 100 mM of NaCl, washed with 10 CV of buffer containing 50 mM of HEPES and 335 mM of NaCl, and eluted with 50 mM of HEPES/890 mM of NaCl buffer. Eluate was concentrated using Amicon Ultra-15 (100 kDa) filter devices (Merck Millipore) followed by 3 washes with PBS and reconcentration. Aliquots of isolated vesicles were lysed with RIPA buffer and diluted in PBS; total protein in samples was measured with a Pierce Coomassie (Bradford) protein assay kit (Thermo-Fisher Scientific, Waltham, MA, USA).