In PBMC co-culture, ELISA assays for prostaglandin E2 (pGE2, Abcam cat. no.: ab133021), tumor necrosis factor alpha-inducible protein 6 (TSG-6, Sigma-Aldrich, cat. no.: RAB1092–1KT), and TGFβ1 (Invitrogen, cat. no.: 88-8350-22) were performed per the manufacturer’s protocols. Additionally, latent TGFβ1 was activated by acidification of the cell culture supernatants, based on manufacturer instructions. Ineffective acidification led to negative absorbance values in several wells in TGFβ1 ELISA assay which were eliminated from the presented data. For the Luminex assay, 50 μL of PBMC culture supernatants were collected and either frozen at −80 °C or immediately analyzed using a human custom ProcartaPlex (11plex, ThermoFisher Scientific, Vienna, Austria) with Luminex MAGPIX. Results were then reported as mean fluorescence intensity.
To measure inflammatory cytokines in spinal cords, the supernatant after cell isolations of spinal cords was harvested. Supernatant solutions (100 μL) were then immediately kept in −80 °C and thawed immediately before performing the Luminex assay. Thawed samples were centrifuged at 10,000 × g for 5 min to extract cells and debris from the solution. We then performed a Luminex assay using Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17-Plex Mouse ProcartaPlex Panel (ThermoFisher Scientific, Vienna, Austria).
To measure inflammatory cytokines in spinal cords, the supernatant after cell isolations of spinal cords was harvested. Supernatant solutions (100 μL) were then immediately kept in −80 °C and thawed immediately before performing the Luminex assay. Thawed samples were centrifuged at 10,000 × g for 5 min to extract cells and debris from the solution. We then performed a Luminex assay using Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17-Plex Mouse ProcartaPlex Panel (ThermoFisher Scientific, Vienna, Austria).