Procartaplex multiplex immunoassay

For quantitation and detection of IFN-γ, IL-1β, IL-10, IL-13, IL-17A and TNF in the supernatant of stimulated cells of the same samples used for AIM, we used a customized ProcartaPlex Multiplex Immunoassay (Invitrogen, Thermo Fisher Scientific). After stimulation and centrifugation of the cells, 100 µl of supernatant was collected in a new 96-well plate and cryopreserved. After thawing the supernatant on ice, the ProcartaPlex assay was performed according to the manufacturer’s instructions. Briefly, after washing the beads twice in a 96-well plate using a magnetic plate washer, 50 µl supernatant was mixed with magnetic beads coupled to antibodies against cytokines and incubated on a microtiter plate shaker (500 rpm) for 120 min at RT. After washing twice, detection antibody was added and incubated for an additional 30 min on a microtiter plate shaker. After washing twice, the concentration of cytokines was analyzed using a Bioplex 200 (Bio-Rad).

Chemokine and cytokine protein concentrations in culture supernatants were assessed after three days of cultivation using a customized mouse ProcartaPlex Multiplex Immunoassay (#EPX010-20440-901, Invitrogen AG, Carlsbad, CA, USA). The assay was conducted following the manufacturer’s protocol and analysis was performed on a MAGPIX™ instrument (Luminex Corporation, Austin, TX, USA).
To determine AQP4-IgG titer of control sera, NMOSD patient sera, and purified IgG samples, an AQP4-IgG sandwich ELISA kit (#RAQP4/96/2R, BioVendor GmbH, Kassel, Germany) was utilized. The ELISA was performed in accordance with the manufacturer’s instructions, and the absorbance was measured at a wavelength of 450 nm on a VarioScan Flash ELISA-reader (Thermo Fisher Scientific, Waltham, MA, USA).

The ProcartaPlex™ Multiplex Immunoassay (EPXR360-26092-901, Invitrogen) was used for measuring cytokine and chemokine levels in the mouse serum after infection. The test was performed according to the manufacturer’s instructions

“Conditioned media” (CM) from MD-macrophages (500µl of RPMI medium without FBS/1. 10⁶ cells) were collected after 24hrs (under normoxia or hypoxia culture conditions). Presence of cytokines in CM (IL1-ß, CXCL8, TNFα, IL-10, PDGF-BB, PAI-1, VEGF-A, IL-18, IL-5) were assayed (undiluted, in duplicates) using multiplex-bead based assays (ProcartaPlex™ Multiplex Immunoassay (Invitrogen) #PPX-16-MXH497N) according to manufacturer’s instructions. Cytokines to be investigated were chosen according to RTqPCR results, and Human cytokine antibody array (proteome profiler™ Bio-techne #ARY022B) (data not shown; analysis performed on 1 control and 2 active sarcoidosis). Cytokine concentrations were automatically calculated or extrapolated if close to the lower limit of detection by the xPONENT software.
Concentrations of active (free) and total (free+latent) TGFß1 in MD-macrophages supernatants were determined by enzyme-linked immunosorbent assays (ELISA) using Duo Set kits (R&D systems #DY240) according to the manufacturer’s instructions.

Proximal colon samples from 14 and 20 weeks of age were stored at –80°C prior to analysis. Samples were sonicated in 300 µl cell lysis buffer (Invitrogen) and refrozen. The thawed suspension was spun for 5 min at 3000 rpm, and the supernatant was assayed for cytokines as per the manufacturer’s recommendations. The ProcartaPlex multiplex Immunoassay (Invitrogen) was used to determine the Th17 panel of cytokines (IFNγ, IL-17A, IL-17E, IL-1β, IL-21, IL-22, IL-6, IL-7R and TNF-α) using Luminex® 200 System. The DC protein assay kit (Bio-Rad laboratories, USA) was used to determine the protein content. Groups that had less than four samples above the limit of the detection were excluded from the analysis. Results were normalized to supernatant protein concentration and are expressed as pg/mg of protein.

FLS were stimulated after overnight attachment to 96 well-plates with recombinant TNF with and without inhibitors for 24 h. Afterwards supernatants were collected and frozen at − 20 °C. The analysis was carried out according to the manufacturer´s user guide (ProcartaPlex™Multiplex Immunoassay, Invitrogen ThermoFisher scientific) using the ProcartaPlex™Multiplex Immunoassay Kit.