Exo flow exosome purification kit

Newborn rats were randomized on postnatal day 1 (P1) into two groups to receive RA or hyperoxia, 85% O₂ as previously described^{21 (link),22 (link)}. On P14, pups were anesthetized, blood samples were collected by right ventricular puncture, and plasmas were obtained. The EVs from an equal volume of plasmas from room air-maintained and hyperoxia-exposed rats were isolated using the appropriate Total Exosome Isolation Kit (ThermoFisher) and resuspended in an equal volume of PBS. A 4 µl from each EV sample were analyzed for particle numbers and size distributions by nanoparticle tracking assay using the Nanosight NS300 system (Malvern Instruments, Malvern, UK) as previously described^{16 (link)}. Nanoparticle concentrations were expressed per ml of plasma. Expression of GSDMD, SPC, CD9 and CD63, in 20 µg of EVs was determined by Western blot analysis as previously described^{22 (link)}. For FACS analyses 25 µg samples of plasma EVs were captured on anti-tetraspanin (CD9, 63 and 81)-conjugated magnetic beads (Exo-FLOW Exosome Purification Kit, Systems Biosciences), stained sequentially with an anti-GSDMD antibody and a FITC-labeled secondary antibody, followed by an PE-labeled anti-SPC antibody, and then analyzed using a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA).

Pregnant Sprague-Dawley rats were purchased from Charles River Laboratory (Wilmington, MA). The following antibodies were used for immunostaining, immunofluorescence staining, Western blot analysis and FACS analysis: rabbit anti-GSDMD and mouse anti-AIF-1 from Novusbio (Littleton, CO); rabbit anti-pro-SPC and mouse anti-CD63 from EDMillipore (Temecula, CA); mouse anti-CD9 antibody from ThermoFisher (Waltham, MA); rabbit anti-Ki67 from Abcam (Cambridge, MA); mouse anti-vonWillebrand factor (vWF) from Dako (Carpinteria, CA); rabbit anti-SPC-PE from Biossusa (Woburn, MA). Total Exosome Isolation Kit was obtained from ThermoFisher. Exo-FLOW Exosome Purification Kit and ExoGlow-Vivo EV Labeling Kit were obtained from Systems Biosciences (Palo Alto, CA). The lipophilic fluorescent dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarocyanine perchlorate (DiI) was purchased from Sigma (Louis, MO). MLE-12 cells were obtained from ATCC (Manassas, VA), pulmonary vascular endothelial cells (PVEC) were obtained from Lonza (Walkersville, MD), and rat fetal neural stem cells (NSC) were obtained from ThermoFisher. The primers for all the qRT-PCR were obtained from ThermoFisher.