Mouse anti cd81

Immunoblotting were performed by using the following antibodies: rabbit anti-M 1:1000 (100-401-A55, Rockland TEBU-BIO); rabbit anti-N 1:3000 (200-401-A50, Rockland TEBU-BIO); mouse anti-S 1:1000 (GTX632604, Genetex); anti-GAPDH antibody coupled to HRP 1:25,000 (G9295, Sigma). For immuno-spotting, rabbit anti-Spike neutralizing Antibody 1:100 (40592-R001, Sino Biological), mouse anti-CD81 1:100 (sc7637, 1.3.3.22, Santa Cruz), fluorescent rabbit Alexa555 and mouse Alexa647-conjugated secondary antibodies 1:2000 (Invitrogen) were used in this study.

Protein content was estimated by 1D-SDS-PAGE/SYPRO^® Ruby protein staining densitometry, as previously described173 (link). For immunoblotting (10 μg), membranes were probed with primary antibodies [mouse anti-TSG101 (BD Transduction Laboratories; 1:500), mouse anti-Alix (Cell Signaling Technology; 1:1000), rabbit anti-CD70 (Abcam; 1:1000), mouse anti-HSPG1 (Abcam; 1:200), rabbit anti-MSLN (Cell Signaling Technology; 1:1000), rabbit anti-FAT1 (GeneTex; 1:2000), mouse anti-CD81 (Santa Cruz Biotechnology; 1:1000), rabbit anti-CALR (Abcam; 1:1000) for 3 hr at room temperature (RT) in 50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20 (TTBS) followed by incubation with either IRDye 800 goat anti-mouse or goat IgG or IRDye 680 goat anti-rabbit IgG (1:15000, LI-COR Biosciences) for 1 hr at RT in TTBS. Immunoblots were visualized using the Odyssey Infrared Imaging System and Image Studio™ Software (v3.0, LI-COR Biosciences, Nebraska USA).

The following antibodies were used: mouse anti-CD9 (Santa Cruz Biotechnology, sc-59140), mouse anti-CD63 (BD Pharmingen, 556019), mouse anti-CD81 (Santa Cruz Biotechnology, sc-555675), mouse anti-β-actin (Millipore, MAB1501), mouse anti-MHC-2 (Cell Signaling Technology, 68258), mouse anti–NK-κB (Cell Signaling Technology, 8242), mouse anti–phosphor-NK-κB (Cell Signaling Technology, 3033), rabbit anti-ANXA1 (Thermo Fisher Scientific, 71-3400), rabbit anti-FPR2 (Novusbio, NLS1878), mouse anti-HT2-280 (Terrace, TB-27AHT2-280), and rabbit anti-SFTPC (Millipore, AB3786). Additionally, the following reagents were used: the Human MACSPlex Exosome Kit (Miltenyi Biotec, 130-108-813), Phorbol 12-myristiate-12 acetate (PMA Sigma-Aldrich, P8139), poly (I:C) (InvivoGen, tlrl-pic), lipopolysaccharide LPS (Sigma‐Aldrich, L2630), dexamethasone (Fujifilm, 041-18861), recombinant ANXA1 (American Research products, 01-2062), and WRW4 (R&D Systems, 2262/1).

Antibodies used were rabbit anti‐p21 (Cell Signaling Technology, 2947), rabbit anti‐p16 (Santa Cruz Biotechnology, sc‐468), mouse anti‐β‐actin (Millipore, Billerica, MAB1501), mouse anti–α smooth muscle actin (Sigma‐Aldrich, A2547), goat anti‐type I collagen (SouthernBiotech, 1310‐01), rabbit anti‐β‐catenin (Cell Signaling Technology, 8480), rabbit anti‐non‐phospho (active) β‐catenin (Cell Signaling Technology, 8814), rabbit anti‐histone H3 (Cell Signaling Technology, 4499), mouse anti‐CD81 (Santa Cruz Biotechnology, 555675), mouse anti‐CD9 (Santa Cruz Biotechnology, sc‐59140), mouse anti‐CD63 (BD Pharmingen, 556019), rabbit anti‐Hsc70 antibody (Proteintech, 10654‐1‐AP) and mouse anti–Tom20 (Santa Cruz Biotechnology, sc‐17764). Hoechst 33242 (Sigma‐Aldrich, H342) and collagen type I solution from rat tail (Sigma‐Aldrich, C3867) were purchased reagents.

EV samples were subjected to standard negative-stain EM. Briefly, a 5 µL of EV sample was placed on a 400-mesh carbon coated copper grid (Electron Microscopy Sciences) that was glow discharged for 20 s. EV samples were allowed to settle on grids for 5 min in a covered glass dish. Each grid was then quickly washed on 2 drops deionized water, wicked with filter paper, and then stained with 1% PTA for 20 s before wicking dry again with filter paper. A phosphotungstic acid staining technique was used to stain and visualize a number of EV preparations from irradiated and non-irradiated MSC. Twelve grids were prepared per condition, and imaged by an operator who was blinded to sample-treatments; images were counted by trained observers in a blinded, random sequence. Images captured using a JEOL JEM-1400 Transmission Electron Microscope (Tokyo, Japan) equipped with a Gata US1000 CCD camera (Pleasanton, CA). For Immunogold labelling, primary human-reactive mouse anti-CD63 (Abcam) and mouse anti-CD81 (Santa Cruz Biotechnology) were used at 10 µg/mL. Colloidal gold (6 nm) conjugated goat anti-mouse secondary antibody was diluted in buffer at 1:20. Negative controls of RPMI and FBS showed no evidence of nonspecific binding.

Total RNA was extracted and purified from EV pellets using the Trizol reagent according to the manufacturer’s protocol (Thermo Fisher). Protein was isolated from retinal tissue using Pierce lysis buffer (Thermo Fisher) with 1% protease inhibitor and 1% phosphatase inhibitor. For EV protein isolation, centrifuged and pelleted EVs were suspended in 200ul PBS. Protein concentrations were obtained using the bicinchoninic acid assay (BCA) assay (Thermo Fisher). Primary antibodies were mouse anti-γ-catenin (Santa Cruz, sc-33634, 1:1000), rabbit anti-actin (Cell Signaling, #8456, 1:5,000), mouse anti-CD81 (Santa Cruz, sc-166028, 1:1000), rabbit anti-CD63 (Novus, nbp2-67425, 1:1000). Blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, Millipore). Band density was quantified using Fiji/ImageJ (NIH).

sEV samples were lysed in non-reducing conditions in Laemmli buffer, heated for 5 min at 95°C, and loaded on mini protean TGX precast 4-20% gradient gels (Biorad) in Tris-glycine buffer for electrophoresis at 110 V for 2 h. Proteins were electro-transferred onto nitrocellulose membranes using the trans-blot turbo transfer system (Biorad) and membranes were blocked with Odyssey blocking buffer (Li-Cor Biosciences) for 1 h. Membranes were then incubated with primary antibodies: mouse anti-CD81 (200 ng/ml, Santa-Cruz), mouse anti-CD63 (500 ng/ml, BD Pharmingen) or mouse anti-CD9 (100 ng/ml, Millipore) overnight at 4°C in Odyssey blocking buffer, followed by incubation with the secondary antibody IRDye 700 goat anti-mouse IgG (Li-Cor Biosciences), for 1 h at room temperature. Membranes were washed three times in TBS 0.1% Tween 20 during 10 min after each incubation step and visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).