Procartaplex multiplex immunoassay kit

Procartaplex multiplex Immunoassay kit was purchased from Thermo Fisher Scientific Waltham, MA, USA to determine and quantify released cytokines in cell culture supernatants. Concentration of IL-6, IL-8, IL-1α, TNF-α protein in the cell culture supernatant were measured after treatment of fpEC with oxysterols according to the user’s manual. The BioPlex-200 suspension array system was used to measure the fluorescence intensity of the samples (Biorad, Hercules, CA, USA). All obtained cytokine concentrations in the supernatant were normalized to total cell protein concentration, which was determined by BCA protein quantification assay.

To determine serum cytokine levels and ALT activity, peripheral blood was taken retro-orbitally upon anesthetization using Isofluran (CP-Pharma) and serum was prepared. IL-36γ was determined by enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s instructions (Cloud-Clone Corp). Levels of eight different cytokines were measured via ProcartaPlex multiplex immunoassay kit (Thermo Fisher Scientific) according to manufacturer’s instructions. ALT activity was determined using a commercially available kit (Hiss Diagnostics GmbH).

A customised Luminex assay was used to measure the following 23 analytes (cytokines/chemokines) in patients’ plasma samples: GM-CSF, IFN-α and IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-10, IL-12p70, IL-13, IL-15, IL-17A (CTLA-8), IL-18, IP-10 (or CXCL10), MCP-1 (or CCL2), MCP-4, MIP-1α, MIP-1β, and TNF-α. The ProcartaPlex Multiplex Immunoassay kit (Thermo Fisher Scientific, PPX-23-MXWCXFA) was used according to the manufacturer’s recommendations. Samples were acquired on Luminex200 Analyser Instrument using the xPONENT basic plus partner analytics. Data were analysed using Invitrogen ProcartaPlex Analysis App on the Thermo Fisher Connect Platform. The two plasma samples from the acute time points that were RT-PCR positive for SARS-CoV-2 were heat inactivated for 30 min at 56°C prior to their use in the Luminex assay. For the matching 3- to 12-month plasma samples, Luminex was performed in parallel on samples that had either been heat inactivated or not, and measurements of all detectable analytes were comparable in these two conditions. Only the data from the non-heat inactivated 3- to 12-month samples was included in Figure 3.

Splenocytes were cultured in RPMI-1640 complete medium (Gibco, containing 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES buffer, 100 IU/mL penicillin, and 100 mg/mL streptomycin) at the concentration of 4 × 10⁶ cells per well and stimulated with 40 μg proteins (BLA, LA–BLA, BLG, or LA–BLG) for 72 h (37 °C and 5% CO₂). Then, the supernatants were frozen at −80 °C until use. All supernatants were thawed simultaneously and assayed to determine the concentrations of secreted cytokines with ProcartaPlex™ Multiplex Immunoassay Kit (Thermo Fisher Scientific, Vienna, Austria), according to the manufacturer’s instructions. These kits allowed the simultaneous quantification of 11 cytokines, including TH1 (IFN-γ, IL-2, and TNF-α), TH2 (IL-4, IL-5, IL-6, and IL-13), TH17 (IL-17A and IL-23), TH9 (IL-9), and Tregs cytokines (IL-10).
The total plasma histamine and serum mMCP-1 levels were measured using commercially available mouse histamine (Demeditec, Berlin, Germany) and mMCP-1 (Neobioscience, Shanghai, China) ELISA kits, in accordance with the manufacturer’s protocol. Individual plasma and serum samples were obtained after challenge and stored at −20 °C before use.

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).

The ProcartaPlex multiplex immunoassay kit was purchased from ThermoFisher Scientific and U-plex array was purchased from Meso Scale Diagnostics (MSD). Blood samples were collected at the end of the experiment from the control and experimental groups. Plasma or serum samples were collected at the time of sacrifice and separated from whole blood following the manufacturer’s instructions. Briefly, for the ProcartaPlex assay, antigen standards were prepared and incubated with magnetic beads. Plasma samples were incubated with the beads for 1–2 h. After washes with the wash buffer, samples were incubated with detection antibody mixture for 30 min at room temperature with shaking. Streptavidin labeled with PE fluorophore was later added to the samples followed by incubation for 30 min. The plate was analyzed further on a Luminex instrument. Samples were subjected to a U-plex array as described^{8 (link)}.

TNF-a and IL-6 levels were measured in the culture supernatants and in the lung homogenates by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. For multiplex ELISA determination, ProcartaPlex multiplex Immunoassay kit was used according to manufacturer instructions (ThermoFisher #EPX360-26092-901). LDH was measured in the culture supernatants using CyQUANT LDH Cytotoxicity kit (Thermo; C20301).