Calnexin 10427 2 ap

Exosomes were isolated by size exclusion chromatography. Briefly, 1 mL of 0.8 μm-filtered serum was diluted 1.5-fold with phosphate-buffered saline and further purified using Exosupur columns (Echobiotech, China) following the manufacturer’s instructions. Fractions were concentrated to 200 μL by 100 kDa molecular weight cutoff Amicon Ultra spin filters (Merck, Germany). The obtained exosome samples were observed by transmission electron microscopy, the particle size distribution was measured by nanoparticle tracking analysis using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany), and their concentration was determined. The exosomes were also identified by western blot analysis using rabbit polyclonal antibody CD63 (sc-5275, Santa Cruz, CA, USA), CD9 (60232-I-Ig, Proteintech, Rosemont, IL), HSP90 (60318-I-Ig, Proteintech, Rosemont, IL), Alix (sc-53540, Santa Cruz, CA, USA), TSG101 (sc-13611, Santa Cruz, California) and calnexin (10427-2-AP, Promega, Madison, Wisconsin). The proteins were visualized on the Tanon4600 Automatic chemiluminescence image analysis system (Tanon, Shanghai, China).

The exosome samples were denatured in 5× sodium dodecyl sulfonate (SDS) buffer before Western blot analysis as previously described. Briefly, 50 µg protein was loaded onto a 10% SDS‐polyacrylamide gel for electrophoresis. The rabbit polyclonal antibodies applied were CD63 (sc‐5275), CD9 (60232‐I‐Ig; Proteintech), HSP90 (60318‐I‐Ig; Proteintech), Alix (sc‐53540), TSG101 (sc‐13611), and calnexin (10427‐2‐AP; Promega). Protein bands were visualized using the Tanon 4600 automatic chemiluminescence image analysis system (Tanon).

The EVs solution is mixed with sodium dodecyl sulfonate (SDS) buffer and subjected to western blot analysis (10% SDS-polyacrylamide gel electrophoresis; 50 µg protein/lane). Under the guidance of protein maker, corresponding bands of CD63, TSG101 and calnexin is cut and transferred to a PVDF membrane. After blocking with BSA, the membrane is incubated with antibodies, including rabbit polyclonal antibody CD63 (sc-5275, Santa Cruz, CA, USA), TSG101 (sc-13,611, Santa Cruz, CA, USA), Alix (sc-53,540, Santa Cruz, CA, USA) and calnexin (10,427–2-AP, Promega, Madison, WI). Finally, the membrane is rinsed to eliminate unbounded antibodies and visualized on the Tanon4600 chemiluminescence image analysis system (Tanon, Shanghai, China).

A total of 10 µL sEV‐enriched fraction was dropped on a copper mesh and incubated at 25°C for 10 min. The sEV‐enriched fraction was negative stained using uranyl acetate for 1 min. Subsequently, the sample was air dried for 2 min under incandescent light. Microphotographs were acquired using a transmission electron microscope (H‐7650, Hitachi Ltd., Tokyo, Japan).
Nanoparticle tracking analysis (NTA) was performed on the sEV‐enriched fractions using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405 nm laser module. The obtained data were analyzed using the corresponding software (ZetaView 8.02.28). To measure the size, concentration, and number of particles in the light‐scattering instrument, samples were diluted by PBS at concentrations between 1 × 10⁷/mL and 1 × 10⁹/mL.
Western blot analysis was performed to identify sEV‐positive (TSG101, sc‐13611, Santa Cruz, CA, USA; CD63, sc‐5275, Santa Cruz; and Alix, sc‐53540, Santa Cruz) and negative (calnexin, 10427‐2‐AP, Promega, Madison, WI, USA) markers. In brief, the sEV supernatant was denatured in 5× sodium dodecyl sulfonate (SDS) buffer and subjected to western blot analysis (10% SDS‐polyacrylamide gel electrophoresis; 50 µg protein/lane) using the above rabbit polyclonal antibodies. Proteins were then visualized on a Tanon 4600 automatic chemiluminescence image analysis system (Tanon, Shanghai, China).