Quantikine elisa kit

IL-6 and IL-8 concentration in Nuli-1 cells exposed to CSE or air in the presence or absence of the NADPH oxidase inhibitor DPI was determined using Quantikine ELISA kits (R&D Systems, Minneapolis, MN). Similarly, IL-6 and CXCXI/KC levels in BALFs collected from mice exposed to CS or air were determined using Quantikine ELISA kits (R&D Systems, Minneapolis, MN). Samples were incubated for 2 h and washed with Quantikine wash buffer as per manufacture instruction. Absorbance was measured at 450 nm with a 540 nm correction, and concentrations calculated per the manufacturer’s instructions.

Cytokine production was measured in the culture supernatants from RPE and RPE^-LC3B cells challenged as described above in the presence or absence of LGM2605 at the concentrations indicated in the Figure legends. Culture supernatants collected at the time points indicated in the figure legends were analyzed by ELISA for IL-1 β (Quantikine Elisa Kit; R&D Systems) and IL-18 (Quantikine Elisa Kit; R&D Systems, #DL180) commercially available kits according to the manufacturer’s instructions [55 (link)]. In each instance, the amount of cytokine present in the supernatant was determined using a standard curve.

Jejunal tissues samples were rinsed in ice-cold saline before being homogenized with phosphate buffer (pH 6–7) utilizing a Mixer Mill MM400 (Retsch, Germany). Centrifugation of the tissue homogenates was performed for 15 min at 10,000× g, 4 °C. Afterward, the supernatant was utilized to quantitively detect the following proinflammatory cytokines per the manufacturer: interleukin-1β (IL-1β) utilizing the rat IL-1β with the Quantikine ELISA Kit (Catalog #RLB00; R&D Systems, Inc., Minneapolis, MN, USA) and tumor necrosis factor (TNF-α) utilizing the rat TNF-alpha with the Quantikine ELISA Kit (Catalog #RTA00; R&D Systems, Inc., USA).

The expression levels of SOST mutants and WT SOST were compared by ELISA and western blot analyses. Each SOST construct was transfected into HEK293A cells using METAFECTENE® PRO (Biontex Laboratories). At 16 h post-transfection, the supernatants from each well (containing secreted SOST) were recovered and centrifuged to remove cell debris. Each SOST mutant and WT SOST in the supernatants were quantified using a Quantikine ELISA Kit (R&D Systems, Catalog #DSST00). The expression levels of WISE mutants were compared to that of WISE WT using an ELISA Kit (MyBioSource, Catalog #MBS9308697).
Since the SOSTΔloop2 mutant was not detected by the corresponding primary antibody of the Quantikine ELISA Kit (R&D Systems), its expression was confirmed by western blotting. Supernatants containing SOSTΔloop2 and WT SOST were prepared as described above. Each sample was concentrated to the same volume, loaded into an SDS-PAGE gel, and visualized by western blot using a monoclonal SOST antibody (Invitrogen, Catalog #MA5-23897, 1:1000 dilution).
Anti-GAPDH antibody (Santa Cruz Biotechnology, Catalog #sc-47724, 1:1000 dilution) was used as a loading control for western blots and anti-His-tag antibody (Cell Signaling, Catalog #2366, 1:1000 dilution) was used for western blots in fluorescence-SEC experiments (Supplementary Fig. 11).

Enzyme-linked immunosorbent assays (ELISA) were used to determine human interleukin (IL)-1 beta and IL-6 (Quantikine® ELISA kit, R&D Systems Inc., Minneapolis, USA) concentrations at 24 h following incubation of Ti60–80, Ti100, Zr2, and ZR75 particles with PMA (5 ng/ml)-stimulated THP-1 cells (DSMZ, Braunschweig, Germany) [5000 cells; in duplicate, binding 24-well plates with the same culture conditions as described above]. THP-1 seeded in empty wells served as cell controls. Recombinant human cytokines (Quantikine® Immunoassay Control Group 1, R&D Systems Inc.) were used as additional positive controls (IL-1 beta, 40–79 pg/ml; IL-6, 80–98 pg/ml; TNF-alpha, 147–265 pg/ml).
Standard curves (x-axis: IL-1 beta/IL-6/TNF-alpha concentrations in pg/ml; y-axis: optical densities) were established according to the instructions given by the manufacturer (Quantikine® ELISA kit). Optical densities of each well were assessed using a microplate reader (Victor X3; Perkin-Elmer, Rodgau, Germany) at 450 nm.