Microbca

Cells were lysed in RIPA buffer for 30 min. After centrifugation, the supernatants were quantified by bicinchoninic acid assay (Micro BCA; Pierce Biotechnology, Rockford, IL). For experiments detecting the secreted ATX protein, the culture medium was concentrated (by approximately 30-fold) using Amicon Ultra 30,000 (Millipore). Protein quantification was conducted and equal amount protein was loaded for each sample. Protein samples were subjected to SDS-PAGE and analyzed as described previously [33 (link)]. Each Western blot analysis was repeated at least three times.

EVs and cells were lysed using RIPA buffer supplemented with Complete^TM mini protease inhibitor (Sigma-Aldrich) at 4 °C for 30 min. Samples were pulse sonicated for 2 s (three times), and were spun at 13,000× g for 30 min at 4 °C. Supernatants were quantified using micro BCA and Pierce BCA (Fisher Scientific) for EVs and cells, respectively. Proteins samples were processed for Western blot and proteomic analyses by mass spectrometry.

Protein attachment on resin disks of 8 mm in diameter and 0.5 mm in thickness (n = 6, for each group at each time point) was measured via a micro-bicinchoninic acid (BCA) approach.^{33 (link),39 (link),56 (link)} Each sample was placed for 2 h in phosphate-buffered saline (PBS). Samples were submerged at 37 °C for 2 h in bovine serum albumin (BSA) solution (Sigma-Aldrich). The protein solution included BSA at 4.5 g ·L⁻¹ concentration, according to a former report.^{57 (link)} The disks were then washed for 5 min with fresh PBS by stirring at 300 r· min⁻¹ (Bellco Glass, Vineland, NJ). The attached proteins were removed in sodium dodecyl sulfate (SDS) at 1% in PBS via sonicating for 20 min. BSA concentration in the SDS solution was evaluated using a protein analysis kit (micro BCA, Fisher Scientific, Pittsburgh, PA).^{33 (link),39 (link),56 (link)} Briefly, a mixture 25 µL of the SDS solution with 200 µL of the BCA working reagent in a 96-well plate was incubated at 60 °C for 30 min.^{33 (link),39 (link),56 (link)} Then, the 96-well plate was cooled to room temperature, and the absorbance at 562 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). Standard curves were prepared using BSA standard. From the concentration of protein, the amount of protein on the sample surfaces was determined.^{33 (link),39 (link),56 (link)}

Poly(ethylene glycol), (PEG: 1 kDa and 4 kDa), methoxy-PEG (550 Da), stannous octoate, ε-caprolactone, poly(vinyl alcohol) (PVA), and lipopolysaccharide were procured from Sigma-Aldrich (St. Louis, MO, USA). L-lactide and hexamethylenediamine (HMDI) were purchased from Acros organics (Morris Plains, NJ, USA). Micro-BCA was obtained from Fisher scientific. Mouse TNF-α, IL-6, and IL-1β ELISA kits were obtained from eBioscience Inc. Lactate dehydrogenase estimation kit and CellTiter 96 AQ_ueous nonradioactive cell proliferation assay (MTS) kit were obtained from Takara Bio Inc. and Promega Corp., respectively. All other reagents utilized in this study were of analytical grade.

A micro bicinchoninic acid (BCA) method was adopted to measure protein adsorption. Briefly, the disks were immersed in bovine serum albumin (BSA; Sigma-Aldrich) solution with a concentration of 4.5 g/L at 37 ℃ for 2 h. The BSA adsorbed on disk surface were detached by sonication and analyzed using a protein analysis kit (micro BCA, Fisher Scientific, Pittsburgh, PA, USA) spectrophotometrically.

Approximately, 25 ml of supernatant from infected and control THP-1 MΦ were collected after 24 h of infection and filtered through 0.2 μm membrane to remove cellular debris, potential membrane fragments from lysed cells, large vesicles (>500 nm) and whole bacteria. The collected material was filtered using an Amicon centrifugal filter unit with a molecular weight cut-off (MWCO) of 100 KDa (EMD Millipore) to 2 ml concentration to remove most of the soluble proteins. The exosome-rich retentate was diluted to 15 ml with PBS and filtered again through the 100 KDa to elute remaining soluble proteins. After this, the retained sample was centrifuged at 18,000 × g for 30 min to pellet larger vesicles. The resultant supernatant was mixed with 400 μl of ExoQuick-TC (SBI) and incubated at 4 °C overnight to precipitate exosomes. After this, the mix was centrifuged at 2,600 × g for 30 min. The pellet-containing exosomes was suspended in 1 ml of PBS and the total protein concentration was measured using the microbicinchoninic acid assay (microBCA, Thermo Scientific). The exosomes were aliquoted (50 μg/vial) and stored at −20 °C until further analysis.

Dissected tissue was homogenized by manual grinding in cold RIPA buffer containing protease and phosphatase inhibitor cocktails (Thermo Scientific) and molecular grinding resin (G Biosciences). Lysates were subjected to three freeze/thaw cycles and clarified by centrifugation at 12,000 × g for 30 min at 4 °C. Protein concentration was determined with a protein assay kit (Micro BCA, Thermo Scientific). Protein lysates (3–5 μg) were separated by polyacrylamide SDS-PAGE gels [15% polyacrylamide for LC3 detection (NB100-2331 from Novus Biologicals) and 7% polyacrylamide for fibronectin (SC-8422 from Santa Cruz) and SQSTM1 (P0067 from MilliporeSigma)] and transferred to PVDF membranes (Bio-Rad). Membranes were blocked with 5% nonfat dry milk in 0.1% Tween-20/TBS and incubated overnight with primary antibodies. The bands were detected by incubation with a secondary antibody conjugated to horseradish peroxidase and chemiluminescence substrate (ECL; GE Healthcare and ECL2, Thermo Scientific). Blots were scanned and analyzed by densitometry using Image J. β-Actin (sc-69879) was used for loading control.