Rat anti human il 6

human IFN-α and interleukin 6 (IL-6) were quantified by ELISA 24 hours after infection. The following antibodies were used: capture: rat anti-human IL-6 (Pharmingen, 554543) or anti-human IFN-α coating antibody (Bender, 2010-10), detection: biotin rat anti-human IL-6 (Pharmingen, 554546) followed by streptavidin-POD conjugate (Roche, 11089153001) or anti-human-IFN-α HRP-conjugate (eBioscience, BM216MSTK). As standards, recombinant human IL-6 (Peprotech, 200-06) or human IFN-α (Peprotech, 300-02A) were used. HRP-substrate solution contained 1 mg/ml OPD (Sigma, P7288) and 0.03% hydrogen peroxide in substrate buffer (38 mM citric acid, 66 mM disodium hydrogen phosphate). Synthetic DNA oligonucleotide with a CpG motive (CpG 2216) (TIB Molbiol) was used as control for TLR activation. Absorbance values were detected with EMax-plate photometer (Molecular Devices) with SoftMaxPro V5-Sofware.

IL6 levels were measured in iAstrocyte supernatants using the Rat antihuman IL-6 and biotin Rat antihuman IL-6 (BD Biosciences, Buccinasco, Milan, Italy) as capture and detection antibodies, respectively. The avidin-conjugated horseradish peroxidase (HRP) and 3,3’,5,5’-tetramethylbenzidine (TMB) microwell peroxidase substrate system (both from Thermo Fisher Scientific, Monza Brianza, Italy) were used for quantification. Colorimetric read-out was analyzed at the Epoch microvolume spectrophotometer system (Biotek, Bernareggio, Italy).