Ab41927

FITC-conjugated lectins from A. integrifolia (Jacalin; FL-1151) and D. stramonium (DSA; FL-1181) were purchased from Vector Laboratories (Burlingame, CA, USA) and FITC-conjugated lectins from A. caudatus (ACA; 21,510,290) were purchased from GlycoMatrix (Dublin, OH, USA). Colloidal gold-conjugated lectins Jacalin-15 nm gold (GP-6301–15), ACA-10 nm gold (GP-8201–10) and DSA-5 nm gold (GP-5701–5) were purchased from EY Laboratories (San Mateo, CA, USA). Lectin inhibitory molecule bovine submaxillary mucin for ACA, galactose for jacalin and chitin hydrolysate for DSA were also purchased from Vector Laboratories. Rabbit polyclonal anti-uroplakin (UP) primary antibody was a kind gift from Prof. Dr. Tung-Tien Sun, Department of Cell Biology, New York University Medical School (Wu et al. 1990 (link)). The following rabbit polyclonal antibodies were also used: anti-flotillin (ab41927, Abcam), anti-clathrin (610,499, BD Transductions), anti-caveolin (ab18199, Abcam) and anti-dynamin 2 (ab65556, Abcam). Goat anti-rabbit IgG AlexaFluor 488 (A11008, Invitrogen) and goat anti-rabbit IgG AlexaFluor 555 (A21428, Invitrogen) secondary antibodies were used. Vectashield mounting medium with DAPI, a nuclei marker, was obtained from Vector Laboratories.

Fludeoxyglucose F18 (¹⁸F-FDG) was produced at the Saskatchewan Centre for Cyclotron Sciences (SCCS), University of Saskatchewan, Canada. PrPc ELISA kit was procured from Biomatik Corporation, anti-rabbit APS (#PA5-17608), anti-rabbit (#7074S) and anti-mouse IgG (#7076S) from Cell Signaling Technology, Dynabeads^® Protein G (#10003D) from Thermofischer Scientific and anti-rabbit flotillin 1 (ab41927), anti-mouse PrPc (ab61409), and anti-mouse CAP (ab182604) antibodies were obtained from Abcam. All other chemicals and reagents purchased were of analytical grade.

Plasma was filtered through 0.2-μm membranes; exosomes were isolated by ultracentrifugation as previously described [12 (link)]; and protein extracts were assessed by Western blotting using antibody against exosomal flotillin-1 (ab41927; Abcam, Cambridge, UK). For validation, the number of particles and the particle size were measured using a nanoparticle-tracking analysis device (NanoSight LM10; Malvern Panalytical, Malvern, UK) coupled to a charge-coupled device camera and a laser emitting a 60-mW beam at 405 nm. Video acquisitions were performed in five recordings of 60 seconds each. At least 1000 particles were tracked in each sample. Nano flow cytometry was carried out using human antibodies against cell surface antigens CD9 (exosome marker) and CD41 (platelet marker) using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA). We used violet side scatter and fluorescent polystyrene beads (Megamix-Plus FSC and SSC; BioCytex, Marseille, France) with known sizes (100, 160, 200, 240, 300, 500, and 900 nm) to identify vesicles smaller than 1 μm. The analysis was performed using CytExpert 2.1 software (Beckman Coulter Life Sciences). Plasmatic concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-α, and transforming growth factor-β were measured in seven patients with sepsis by enzyme-linked immunosorbent assay.

27-Hydroxycholesterol was provided by American Santa Cruz, and 27-OHC (10 mg) was completely dissolved in a suitable amount of ethanol, then divided equally in centrifuge tubes, with nitrogen gas blowing dry, and finally preserved at −80°C. The 27-OHC working solution contains 0.16% ethanol (v/v). Filipin III was from Cayman Chemical (#70440, Ann Arbor, MI, United States). Primary antibodies for Western blot included mouse anti-ABCA1 (Abcam, ab66217), rabbit anti-ABCG1 (Abcam, ab52617), mouse anti-Caveolin-1 (Abcam, ab17052), rabbit anti-Apolipoprotein E (Abcam, ab52607), mouse anti-LDLR (Millipore, MABS26), rabbit anti-ACAT1 (Abcam, ab168342), rabbit anti-CYP46A1 (Sigma–Aldrich, SAB2100523), mouse anti-KDEL (Santa Cruz, sc-58774), rabbit anti-flotillin (Abcam, ab41927), rabbit anti-SR-B1 (Abcam, 52629), mouse anti- N Cadherin (Abcam, ab98952), mouse anti-Aβ (Abcam, ab126649), and rabbit anti-LRP1 (Abcam, ab92544). Goat anti-mouse (#14709) and rabbit (#7074) biotinylated secondary antibodies were from Cell Signaling Technology. Antibodies for immunofluorescence included anti-CYP46A1 (Abcam, ab82814), goat anti-mouse (Abcam, ab150120), and goat anti-rabbit (Abcam, ab96899).

EVs were isolated from 500 µL of the plasma using qEV Original/70 nm (Izon, Christchurch, New Zeland) size-exclusion chromatography (SEC) columns, according to the manufacturer’s instructions. The size and concentration of collected EVs were analyzed by Nanoparticle Tracking Analysis (NTA) (NanoSight LM10, Malvern Instruments Ltd., Malvern, UK). The EVs were checked by transmission electron microscopy (TEM), as previously described [20 (link)]. EV protein expression was analyzed by western blot, as previously described [20 (link)], using an anti-flotillin-1 (1:10,000 dilution, ab41927, Abcam), anti-PD-L1 (1:1000 dilution, 13684, Cell Signaling, Danvers, MA, USA) and anti-B7-H3 antibody (1:1250 dilution, MAB1027-100, R&D Systems Biotechne, Minneapolis, MN, USA). Anti-rabbit (ECL-antirabbit IgG, NA934V, Amersham, dilution: 1:2500) or anti-mouse (ECL-anti mouse IgG, NA931V, Amersham, 1:2000 dilution) secondary antibodies were used.