Milliplex map human complement panel

Complement components in the cellular supernatants after 11–14 days of cultivation were quantified using the MILLIPLEX MAP Human Complement Panel (Merck, #HCMP1MAG-19K, #HCMP2MAG-19K) in accordance to the manufacturer’s protocol. The read out of the multiplex assay was performed in a Magpix instrument (Luminex, Austin, TX, USA).

Properdin levels in cell culture supernatants were determined using a sandwich ELISA, as previously described [37 (link)]. C3 quantification was performed using a newly developed C3 ELISA (cat# HK366-01; Hycult Biotech, Uden, Netherlands) according to the manufacturer’s protocol. CFH was quantified in an in-house ELISA with mouse anti-CFH monoclonal antibody (BioRad, Feldkirchen, Germany) as a capture antibody and goat anti-CFH polyclonal antibody (Merck, Darmstadt, Germany) as a detection antibody (Supplementary Materials, Table S1). A comparison of properdin, C3, and CFH levels in supernatants of lower and higher ARPE-19 passages was performed using a MILLIPLEX MAP Human Complement Panel (Merck, Darmstadt, Germany). Interleukin (IL)-1β und IL-6 concentrations were determined according to the protocol of a custom ProcartaPlex^® multiplex immunoassay kit (ThermoFisher, Waltham, MA, USA). IL-8 was analyzed using xMAP technology, anti-IL-8 beads (cat# 171-BK31MR2; BioRad, Feldkirchen, Germany), and anti-IL-8 biotin (cat# 171-BK31MR2; BioRad, Feldkirchen, Germany) according to the manufacturer’s protocol. The readout of the multiplex assays (IL-1β, IL-6, and IL-8) was performed using a Magpix instrument (Luminex, Austin, TX, USA). Vascular endothelial growth factor (VEGF)-α concentrations were determined using a human VEGF Quantikine ELISA Kit (R&D system, Minneapolis, MN, USA).