Cd81 monoclonal antibody

The proteins of suspensions OFs, exosomes, and tissues were obtained and quantified by the BCA method (Beyotime, Shanghai, China). Protein lysates were transferred to the PVDF membrane by 8% SDS-PAGE (Beyotime Biotechnology, China). The PVDF membrane was cleaned with TBST and sealed with 5% skim milk powder for 2 h. The cut film was incubated overnight in the primary antibody dilution solution and 2 h in the secondary antibody dilution solution. Finally, the film was scanned. The main antibodies used included HRP-binding GAPDH monoclonal antibody (ProteinTech, Wuhan, China), AKT polyclonal antibody (ProteinTech, China), NFκB P65 polyclonal antibody (ProteinTech, China), Rabbit Anti-phosphorylated NFκB P65 (Ser276) Antibody (Bioss, Beijing, China), Rabbit Anti-phosphorylated AKT (Ser473) antibody (Bioss, China), IL-1α polyclonal antibody (ProteinTech, China), ICAM1 polyclonal antibody (ProteinTech, China), CD9 monoclonal antibody (ProteinTech, China), TSG101 polyclonal antibody (ProteinTech, China), CD81 monoclonal antibody (ProteinTech, China), Goat Anti-mouse IgG (H+L) (ProteinTech, China), Goat anti-rabbit IgG (H+L) (ProteinTech, China), and glucocorticoid receptor polyclonal antibody (ProteinTech, China).

The concentration of EVs was estimated based on the total protein content, determined using a bicinchoninic acid (BCA) protein assay kit (Cat. No. E-BC-K318-M, Elabscience). Nanoparticle tracking analysis (NTA) with a ZetaView PMX 110 (Particle Metrix) was used to analyze the distribution and size of EVs, while Hitachi H-7650 transmission electron microscope (TEM; Hitachi) was used to identify the morphologies of EVs. To assess the effectiveness and stability of each batch of EVs, cellular functional tests and NTA analysis were employed. Flow cytometry on a FACSCANTO II (BD Biosciences) was employed to resolve EV surface marker proteins, including CD63, CD81, and TSG101, as previously described [48 (link), 49 (link)], and FlowJo software (Tree Star Inc) was used to analyze the results. The CD63 polyclonal antibody (Cat. No. 25682-1-AP, Proteintech), CD81 monoclonal antibody (Cat. No. SC7637, Santa Cruz Biotechnology), TSG101 polyclonal antibody (Cat. No. 14497-1-AP, Proteintech) were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rabbit IgG (Cat. No. 111-545-144, Jackson ImmunoResearch) and Alexa Fluor 594-conjugated donkey anti-mouse IgG (Cat. No. 715-585-151, Jackson ImmunoResearch) were used as secondary antibodies.