Quantifying Antimicrobial Molecules in Platelet Supernatants

Supernatants collected from platelets treated with LL-37 or the control condition (Section 4.2) and the secretion of BPI, MPO, HNP1 (R & D System), and Azu (Sino Biological, Beijing, China) were quantified by ELISA (enzyme-linked immunoadsorbent assay) using an optical density. A capture antibody was used for each antimicrobial molecule and incubated overnight. Subsequently, blocking was performed using bovine albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) for one hour. Next, 100 µL of the sample was placed in each well per condition and left to incubate for two hours. Afterwards, the wells were washed with PBS buffer (Phosphate buffered saline) three times, and a detection antibody was added, followed by a secondary antibody coupled to HRP (rabbit peroxidase), according to the instructions from each supplier of the ELISA Kits. Readings were performed at 450 nm using a microplate reader (Model 4300, Awareness Technology Inc. Chromate, Palm City, FL, USA) with the Leica Suite 345 X software application (LAS-X, Leica-Microsystems, Wetzlar, Germany).