Cytokine antibody array

Quantification of 13 cytokines in patient urinary EV specimens was performed using a custom antibody array (RayBiotech, SO2-AAH-CYT-CUST) according to the manufacturer’s instructions. Briefly, we prepared 40 µg of urinary EV protein per patient in 100 µL of DPBS. Triton X-100 was added to a final concentration of 1%, and the samples were vortexed and incubated for one hour at room temperature to permeabilize the EV membranes and allow the release of cytokines. EV samples were then diluted to 1 mL in DPBS, and protease inhibitor was included to make a 1× working concentration (Pierce, A32953). Following a 30 min block, membranes were incubated with EV samples overnight at 4 °C. Membranes were photographed with a Bio-Rad ChemiDoc XRS+, and the images were analysed with ImageJ. Background subtraction was performed on each image using a rolling ball radius of 50 pixels, and the integrated density of each dot in the blots was measured with the MicroArray Profile plugin. Normalisation of samples using the positive control signal intensities was completed with the RayBiotech C-Series analysis tool.