Lxsahm 02

iRPE on transwells were treated with CI-HS or CC-HS for 48 h. Media from the apical and basal sides of the transwells were collected in 1.5-mL Eppendorf tubes and centrifuged at 17,243 × g and 4 °C for 15 m. The supernatants were transferred into fresh 1.5-mL Eppendorf tubes, immediately placed in dry ice, and stored at −80 °C. ELISA kits for cytokines IL-8 (LXSAHM-02, R&D) and IL-18 (LXSAHM-02, R&D) were used per the manufacturer’s protocol. Samples were diluted fivefold in the diluent buffer. ELISA kits for complement proteins CFH (A039, Quidel), C3a (A031, Quidel), and C5a (A021, Quidel) were used per the manufacture’s protocol. Samples for the CFH and C3a assays were diluted 50-fold, and samples for the C5a assay were diluted fivefold. ELISA plates were analyzed using a TECAN SAFIRE plate reader. Results account for different apical and basal media volumes used for transwells.

Human Magnetic Luminex Assays (LXSAHM-15, LXSAHM-08, and LXSAHM-02) were purchased from R&D Systems (Minneapolis, MN). Magnetic COOH beads, amine coupling kits, and Bio-Plex Pro Reagent kits were purchased from Bio-Rad Laboratories (Hercules, CA). NHS and Sulfo-NHS, EDC, EZ-Link^TM Sulfo-NHS-Biotin, and Zeba^TM Spin Desalting Columns were purchased from Thermo Scientific (Rockford, IL). Agarose bound Aleuria Aurantia Lectin (AAL) was purchased from Vector Laboratories (Burlingame, CA). Pierce™ BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA).

Twenty-nine parameters were analyzed in serum samples, by magnetic bead assays or ELISA: Human XL Cyt Disc Premixed Mag Luminex Perf Assay Kit (CCL2/MCP-1, CCL4/MIP-1β, RANTES/CCL5, CCL11/Eotaxin, CCL19/MIP-3β, CCL20/MIP-3α, CD40Ligand, Fractalkine/CX3CL1, CXCL10/IP-10, EGF, Flt-3 Ligand, granzyme B, IL-1RA, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-15, PD-L1/B7-H1, TNF-α, TRAIL/TNFSF10, and VEGF) (R&D Systems, #FCSTM18-22). For the Human Magnetic Luminex Assay (CD163, ST2, and CD14, LBP) (R&D Systems, LXSAHM-02), plates were prepared according to the manufacturer’s recommendations and read on a Bio-Plex 200 System™ version 6.1. Amphiregulin (#DAR-00), FABP2/IFABP (#DFBP20), and haptoglobin (#DHAPGO) were determined with Human Quantikine ELISA Kits (R&D Systems), according to the manufacturer’s instructions.