Costar eia ria plates

Cytokine levels in cell culture supernatants were measured by sandwich ELISA. Microtiter plates (Costar EIA/RIA plates, Corning Inc.) were coated with a cytokine-specific antibody. Expression levels of IL-6, IL-10, and IL-12p40 were measured according to the manufacturer’s instructions (OptEIA Sets, BD Biosciences). TNF-α level was measured according to the manufacturer’s instructions (ELISA Ready-Set-Go, eBioscience). In all cases, 10% FCS in PBS was used as a blocking solution. TMB substrate solution (BioLegend) was used to develop a colorimetric reaction, which was stopped with 2 M sulfuric acid. Optical density was measured at 450 (570) nm using a microtiter plate reader (PowerWaveX, Bio-Tek Instruments).

hrRNASET2 binding to actin was quantified as modified from Liu et al. [22 (link)]. Human platelet actin (5 ug/ml) (Cytoskeleton, Denver, CO, USA) was diluted in carbonate-bicarbonate buffer (Sigma-Aldrich, St Louis, MO, USA), pH 9.5, and coated directly onto 96-wells COSTAR EIA/RIA plates (Corning, NY, USA) overnight at 4°C. The plates were then blocked with 5% (w/v) BSA in PBS containing 0.25% Tween-20 (PBST), at room temperature for 1 h, and subsequently incubated with hrRNASET2, at 1:2 serial dilutions in PBST, overnight at 4°C. Plates were washed three times with TBST, under continuous shaking for 10 min each, at room temperature and then incubated with rabbit anti-RNASET2 polyclonal affinity pure antibodies (GENEMED SYNTHESIS, San Antonio, TX, USA), diluted 1:500 in PBST at 37°C for 1 h. After three washing as described above, 0.8 μg/ml peroxidase-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in TBST was added and incubated under the same conditions. Plates were then washed twice with TBST and once with TBS, as described above. 1-Step Ultra TMB-ELISA solution (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA) was then added and optical absorbance was detected at 650 nm with an Infinite F50 multidetection microplate reader (Tecan, Grödig, Austria).

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against mRo60_316-335 peptides. The SSA peptides (10 mg/ml in 0.5 M Na₂CO₃ buffer; pH 9.6) were absorbed onto Costar EIA/RIA Plates (Corning Incorporated, Corning, NY, USA), washed and blocked with 3% BSA in PBS with 0.05% Tween-20 (PBS-T), incubated with the respective mouse sera (1:200), and further washed with PBS-T. Bound antibodies were detected using peroxidase conjugated goat anti-mouse IgG antibodies (Sigma, USA) and tetramethylbenzidine (Solarbio, Beijing, China). Concentrations of IFN-γ, IL-17A, IL-4, and IL-10 in mouse sera were determined by commercially available ELISA kits (Peprotech, USA) according to the manufacturer’s protocols. The detection range of the assay for IFN-γ, IL-17A, IL-4, and IL-10 was 7.5–1,000 pg/ml, 7.5–1,000 pg/ml, 40–5,000 pg/ml, and 15–2,000 pg/ml, respectively. All sera were diluted 1:3 before quantification.

Anti-CII antibodies in serum were measured by sandwich ELISA as described previously (Kwaśny-Krochin et al. 2002 (link)). In brief, plates (Costar EIA/RIA plates, Corning Incorporated, USA) were coated overnight with native chicken CII (5 μg/ml). Mouse serum samples diluted in PBS were applied to antigen-coated wells for 1 h at room temperature followed by biotin-conjugated antibodies against mouse IgG (Sigma-Aldrich, Germany), IgG1 (MP Biomedicals, USA) or IgG2a (Southern Biotech, USA). Horseradish peroxidase-conjugated streptavidin (Vector, USA) was used as detection reagent and developed with TMB Substrate Solution (Thermo Fisher Scientific, USA). The reaction was stopped with 3 M H₂SO₄ (POCH, Poland). The optical density of each sample was measured at λ = 450 nm. The antibody levels were expressed in arbitrary ELISA units (AU) calculated from the titre of anti-CII immunoglobulins at OD = 0.1 × 10.^–2

Mouse IFN-γ ELISA Ready-SET-Go! Kit (Invitrogen, Thermo Fisher Scientific, USA) was used and the assay was performed according to the manufacturer’s instructions. Shortly, Costar EIA/RIA plates (Corning Incorporated, USA) were coated with anti-mouse cytokine antibodies overnight at 4 °C. After blocking the plates, serial dilutions of mouse IFN-γ standards and diluted cell culture supernatants were applied to the plates and incubated overnight at 4 °C. Then biotin-conjugated anti-IFN-γ antibodies were added for 1 h (room temperature), followed by avidin-HRP and TMB as detection reagent. The optical density was read at 450 nm.

Cytokine levels in cell culture supernatants were measured by sandwich ELISA. Microtiter plates (Costar EIA/RIA plates, Corning) were coated with a cytokine-specific antibody. The expression levels of IL-6, IL-10, and IL-12p40 were measured according to the manufacturer’s instructions (OptEIA Sets, BD Biosciences). TNF-α level was measured according to the manufacturer’s instructions (ELISA uncoated kits, Invitrogen, Waltham, MA, USA). In all cases, 10% FBS in PBS was used as a blocking solution. TMB substrate solution (Invitrogen) was used to develop a colorimetric reaction, which was stopped with 2 M sulfuric acid. Optical density was measured at 450 (570) nm using a microtiter plate reader (PowerWaveX, Bio-Tek Instruments, Winooski, VT, USA).