Exoquick tc ultra kit

Exosomes were derived from human brain microvascular endothelial cells (hBMVEC) using ExoQuick-TC ULTRA kit (Systems Biosciences) according to the protocol. Exosomes’ surface markers, size, morphology, and concentration were characterized using flow cytometry, DLS, TEM, and protein assays, respectively. Next, exosomes were surface modified with EDC/NHS chemistry and subsequently conjugated with tPA. For the conjugation reaction, 200 μL of 50 μg/mL exosomes was reacted with 2 μL of 100 mM EDC and 2 μL of 100 mM NHS for 20 min, and subsequently, 20 μL (1 mg/mL) of tpA was added and stirred overnight at 4 °C. The exosomes were then centrifuged and resuspended in 220 μL HEPES buffer with a pH of 7.5. We used HEPES buffer for the reactions, as it has been widely used in tPA formulations [54 (link),55 (link),56 (link)]. To quantify the tPA conjugation to the exosomes, FITC (fluorescein isothiocyanate) or rhodamine-labeled tPA was used for the exosomes’ conjugation and quantified by the FITC or rhodamine fluorescence using an M3 SpectraMax plate reader. The particles’ size, morphology, and tPA protein conjugation were characterized by DLS, TEM, protein assay, and tPA activity assay.

The ExoQuick-TC ULTRA kit (System Biosciences, Palo Alto, CA) was used, according to the manufacturer’s instructions, in order to isolate mf-derived EVs after 24 hours of incubation. A NanoSight NS300 and NTA software were used to assess the average size and concentration of the particles in the purified preparation [31 (link)].

Human bone marrow derived mesenchymal stem cells (hBMDSCs) were obtained from ATCC (American Type Culture Collection, Manassas, Virginia, USA). The cells were grown in T75 tissue culture flasks with mesenchymal stem cell basal medium (ATCC) with 10% fetal bovine serum, and 1% antibiotic-antimycotic. Cells were allowed to reach 70–80% confluence for 48 hr and then cells were maintained in serum free media for 24 hr before supernatant was collected. EVs were collected from the supernatants of h-BMDSCs according to the MISEV2018 guidelines. EVs were isolated and purified from the supernatants collected, using ExoQuick-TC® ULTRA kit (Systems Biosciences, Palo Alto, CA, USA) according to the manufacturer protocol. We also used ultracentrifugation method for the isolation of EVs. Both methods provided a similar yield (Supplemental Fig. 1).