Cw 50r 1.0 culturewell gasket

Amino group-functionalized glass slides (NH₂-glass, Nanocs Inc.) were ultrasonically cleaned in methanol for 5 min, thoroughly flushed with deionized water, dried under an N₂ stream, and then the NH₂-functionalized glass was covered with a 50 well polydimethylsiloxane membrane (CW-50R-1.0-CultureWell Gasket, Grace Bio-Labs Inc.). Slides were filled with 10 μL/well of 12.5 μg/mL anti-human CD81 antibody (R&D systems, clone #454720) in 5 mM EDC/PBS and incubated at room temperature for 2 h in a moist chamber, aspirated and filled with 10 μL/well of 5 % BSA/PBS (pH 7.0) and incubated at room temperature for 4 h, then PBS (pH 7.0) washed and aspirated three times prior to loading these wells with analysis samples. Sample wells were filled with 5 μL/well of plasma samples (diluted 40 with pH 7.0 PBS) or cell culture EV samples, incubated at room temperature for 4 h in a moist chamber, washed three times with PBST, three times with PBS and then filled with 7 μL/well of AuS and AuR PBST probe solution (4 × 10⁻¹¹ M for each particle) and incubated for at room temperature for 1 h, after which the PDMS cover was removed, the slide was washed three times with PBST and three times with PBS, then fitted with a cover slip and imaged by dark-field microscopy (DFM).