Nanoparticle tracking analysis

Cells used for exosome isolation were grown in exosome-depleted FBS (System Biosciences; Mountain View, CA). Exosomes were isolated from cell culture media according to the manufacturers instructions using the Total Exosome Isolation (from cell culture media) reagent (Invitrogen-Life Technologies). Briefly, IMR32 cells were seeded at 1.25×10⁵ cell /well in a 24 well plate, 24 hours after seeding cells were treated with either SFN, TBOOH, or DMSO for 24 ours. 48 hours after seeding (24 hours after adding treatment) the cell culture media was collected and centrifuged at 2000xG for 20 minutes and a final cell count was obtained (cell/ml) for each individual well. The cell-free supernatant containing the exosomes was transferred to a clean tube and 0.5 volumes of the Total Exosome Isolation reagent was added. Samples were thoroughly mixed and stored at 4°C overnight, and then finally samples were centrifuged at 10,000xG for 1 hour. Supernatant was discarded and the exosomes were resuspended in 50μL phosphate buffered saline. Samples were analyzed for exosomes (30-100nm) using a NanoSight and the Nanoparticle Tracking Analysis (Malvern Instruments, Worcestershire, UK). Exosome counts (particles/mL) were normalized to cell count (cells/ml) to obtain exosomes/cell.

Differential centrifugation and filtration methods were adapted for isolation of plasma microvesicles, exosomes, and small apoptotic bodies (sABs).^{15 ,16 (link)} Briefly, blood collected using 3.8% sodium citrate solution was slowly centrifuged for 10 minutes (4°C; 300g). Then, 250 μL of plasma from mice and humans and 700 μL from rats, respectively, was centrifuged twice for 20 minutes (1500g; 4°C). Platelet-free plasma was either filtered (0.8 μm mesh) and centrifuged at 12 200g (40 minutes; 4°C) for isolation of microvesicles or 16 100g for 20 minutes for collection of microvesicles+sABs. In both cases, the pellet was either resuspended in PBS or RIPA (radioimmunoprecipitation assay) buffer and supernatant was filtered (0.2 μm mesh) and ultracentrifuged at 100 000g (60 minutes; 4°C) for exosome isolation. EVs were diluted in double-filtered PBS for determination of size and concentration by Nanoparticle Tracking Analysis (NanoSight; Malvern Panalytical) or resuspended in RIPA buffer plus protease and phosphatase inhibitor cocktail for Western blot. EV morphology was assessed by transmission electronic microscopy.

The total and platelet derived microvesicle populations were isolated and quantified as previously described.^{18 (link),20 (link),21 (link)} Briefly, whole blood from a cardiac puncture was anticoagulated with 10 % citrate and centrifuged at 450g for 10 minutes. The supernatant was subsequently collected and centrifuged again at 10,000g for 10 minutes at 4°C. The cell- and platelet-free supernatant was then diluted with Roswell Park Memorial Institute media and stained with CD41 antibody (0.2 mg/ml) (clone MWReg30, BD Biosciences, San Jose, CA)). Nanoparticle Tracking Analysis (Malvern Instruments Ltd, Worcestershire, United Kingdom) was then used to quantify total and CD41+ microvesicle concentrations. Microvesicles were quantified at 24 hours following injury based on previous work from our laboratory that demonstrated platelet derived microvesicles to be most elevated at this timepoint.^{20 (link)}

Microvesicles were isolated as previously described¹⁹ . In brief, bronchoalveolar (BAL) fluid was collected from sham and post burn day 1 (PBD1) mice and centrifuged at 450 × g for 10 minutes at room temperature to form a cell pellet. Supernatant was collected and centrifuged at 10,000 × g for 10 minutes at room temperature to remove any residual cells. Supernatant was then centrifuged at 25,000 × g for 30 minutes at room temperature to pellet MVs. MV pellets were re-suspended in 1 ml of Roswell Park Memorial Institute media (RPMI) and 1:100 MV dilutions were quantified using Nanoparticle Tracking Analysis (Nanosight, Malvern Instruments Ltd, Worcestershire, UK)^{22 (link)}.