Il 1 beta mouse uncoated elisa kit

Utilizing Chemray 800 (Shenzhen, China) and the manufacturer's instructions, serum concentrations of the enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected to evaluate mouse liver function. Employing commercial enzyme-linked immunosorbent assay (ELISA) kits (IL-6 Mouse Uncoated ELISA Kit, 88-7064-88; IL-1 beta Mouse Uncoated ELISA Kit, 88-7013-88; and TNF alpha Mouse Uncoated ELISA Kit, 88-7324-22 from Invitrogen) in accordance with the manufacturer's protocol, the inflammatory state was determined by quantifying serum cytokines.

Conditioned media were collected and preserved at −70 °C until analysis. Levels of TNFα and interleukin 1ß (IL1ß) were measured using the Mouse TNF alpha uncoated ELISA kit and IL-1 beta Mouse Uncoated ELISA Kit, respectively (Invitrogen, Thermo Fisher Scientific). Samples were analyzed in duplicate. TNFα results were obtained in ng/mL and IL1β results in pg/mL.

Sera cytokines were measured by Luminex at the University of Virginia Flow Cytometry Core, or by ELISA (Thermo Fisher Scientific). Cytokine measurement on tissue homogenate was assessed using ELISA (Mouse IFN gamma Uncoated ELISA (Invitrogen, #88-7314); Mouse TNF alpha Uncoated ELISA (Invitrogen, #88-7324); IL-1 alpha Mouse Uncoated ELISA Kit (ThermoFisher #88-5019-88); eBioscience Mouse IL-6 ELISA Ready-SET-Go! Kit (Fisher Scientific #50-112-8863; IL-1 beta Mouse Uncoated ELISA Kit (Thermo Fisher Scientific #88-7013-88).

At the end of the priming incubation, the medium containing LPS was replaced with growth medium containing 0 or 20 μM Z-WEHD-FMK (catalog no. FMK002; R&D Systems), an irreversible caspase-1 inhibitor, and the cells were incubated 1h under cell culture conditions. Co²⁺ (6 to 48 ppm final concentrations), Cr³⁺ (100 to 500 ppm), Ni²⁺ (12 to 96 ppm), nigericin (5 μM; positive control), or cell-culture grade water (ion solvent used as the negative control) was then added to the culture supernatants and the cells were incubated an additional 18 to 24h. At the end of the incubation, supernatants were collected, centrifuged at 300 g for 10 min at 4°C, snap-frozen in liquid nitrogen, and stored at -80°C for later analysis of IL-1β release by enzyme-linked immunosorbent assay (ELISA).
Freshly thawed culture supernatants were gently mixed and concentrations of IL-1β were measured by ELISA using a commercial kit (IL-1 beta Mouse Uncoated ELISA Kit; Thermo Fisher Scientific), as per the manufacturer’s instructions. Absorbance measurements were performed at 450 nm using a hybrid microplate reader (Synergy™ 4; BioTek, Winooski, VT). Absorbance at the reference wavelength of 570 nm was subtracted from the measurements. The nominal minimum concentration of IL-1β detectable by ELISA was 8 pg/mL, as per the manufacturer’s specifications.

ELISA using the mouse IL-6 Uncoated ELISA Kit (Invitrogen, Carlsbad, CA, USA), mouse IL-1 beta Uncoated ELISA Kit (Invitrogen), and mouse TNF alpha Uncoated ELISA Kit (Invitrogen) was performed using serum in accordance with the instructions of the manufacturers. The limits of detection for each analyte were as follows: mouse IL-6, 4 pg/mL; mouse IL-1β, 8 pg/mL; mouse TNF-α, 8 pg/mL.