Amicon ultra 15 100 kda filter devices

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).

EVs were isolated from HEK-293T cell complete cell culture media as described previously [25 (link)]. Before isolation, HEK-293T cells were cultured in complete DMEM media supplemented with FBS depleted of animal EVs by ultrafiltration using Amicon Ultra-15 100 kDa filter devices (Merck Millipore, Darmstadt, Germany), as shown in [26 (link)]. In brief, conditioned media were centrifuged at 2000× g for 10 min and then at 10,000× g for 10 min to remove cell debris and large EVs followed by anion-exchange chromatography using MacroPrep DEAE Media anion-exchange resin (BioRad, Hercules, CA, USA). The column was washed with 100% buffer A (50 mM HEPES, 100 mM NaCl) followed by stepwise washing with 95% buffer A/5% buffer B (50 mM HEPES, 2 M NaCl)—5 column volumes (CV), 90% buffer A/10% buffer B—10 CV. The fraction containing EVs was eluted with 60% buffer A/40% buffer B and the column was then washed with 100% buffer B. The eluate was concentrated using Amicon Ultra-15 (100 kDa) filter devices followed by 3 washes with PBS and 1 more concentration step.