Sector imager 600

Prototype ultrasensitive electrochemiluminescence assays (Meso Scale Diagnostics, Rockville, MD) were used for intact EV surface marker detection. Three multiplexed assay panels were used in this study (as listed in Supplementary table 1). 5 μL of each bdEV sample was diluted 1 to 40 in MSD diluent 52 and samples were added to assay plates with capture antibody arrays and shaken continuously at RT during the EV capture step. Panel 1, comprising antibodies targeting relatively abundant surface markers, was incubated for 1 hour, while the remaining panels, targeting lower-abundance markers, were incubated for 4 hours to improve sensitivity. EVs captured by each antibody were detected using prototype MSD S-PLEX^® detection reagents with a cocktail of detection antibodies targeting CD63, CD81, and CD9. Assay plates were read with MSD GOLD^™ Read buffer B on an MSD^® SECTOR 600 imager. ECL signal from a DPBS blank on each assay spot and ECL signal from each bdEV sample on an isotype-control capture spot were subtracted consecutively from the signal of each corresponding assay to account for non-specific binding of detector antibodies and the EVs in the sample, respectively.

Detection of circulating Klotho levels from mouse blood samples was performed by using an ELISA assay on the MSD platform. Standard-bind MSD plates (#L15XA-1, MSD) were coated with mouse Klotho capture antibody (#AF1819, R and D) at a final concentration of 4 µg/mL in PBS for 1 hr at room temperature. Subsequently, plates were washed three times using wash buffer (PBS + 0.05% Tween-20) at 300 µL/well followed by an incubation with 3% blocker A solution (R93BA-2, MSD) for 1 hr at room temperature. Dilutions of serum samples and murine recombinant Klotho standard (#1819 KL-050, R and D) were prepared in 1% blocker A solution and added to the MSD plate in a final volume of 25 µL/well after the plate was washed three times. Samples were incubated for 1 hr at room temperature followed by three washing steps. Detection antibody (#BAF1819, R and D) was diluted to 1 µg/mL in 1% Blocker A solution in PBS and SULFO-tag labeled streptavidin (#R32AD-5, MSD) was diluted to 0.5 µg/mL in 1% Blocker A in PBS. Both dilutions were added to the plate simultaneously (25 µL/well each) and incubated for 1 hr at room temperature. After three washing steps, 1x Read Buffer (#R92TC-2, MSD) diluted in water was added at 150 µL/well. Electrochemiluminescence was detected in the MSD Sector Imager 600.