Mouse il 1β quantikine elisa kit

Cell culture supernatants were collected after centrifuging at 300 × g for 20 min. Concentrations of TNF-α, IL-6, and IL-1β were measured using the Quantikine Mouse TNF-α ELISA Kit (R&D Systems, Minneapolis, MN, USA), Quantikine Mouse IL-6 ELISA Kit (R&D Systems), and Quantikine Mouse IL-1β ELISA Kit (R&D Systems), respectively [13 ]. Since the detection limits of TNF-α, IL-6, and IL-1β using ELISA are 10.9–700 pg/ml, 7.8–500 pg/ml, and 7.8–500 pg/ml, respectively, the supernatants were diluted 50 times before TNF-α and IL-6 concentrations were measured.

The RAW 264.7 cells were plated at 5 × 10⁵ cells/mL and stimulated with LPS (200 ng/mL) in the presence or absence of terrein (1, 3, 10, or 30 μM) for 24 h. The culture supernatants were collected and the amount of cytokine was determined using a mouse IL-1β Quantikine ELISA kit or mouse IL-6 Quantikine ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.

ELISAs were performed on culture supernatants using the Mouse IL-12/IL-23 p40 Non-allele-specific and Mouse IL-10 Quantikine ELISA kits (R&D Systems) according to manufacturer’s instructions. Cytokine arrays were performed on 1 ml of supernatant from the co-cultures using the Mouse Cytokine Antibody array, Panel A (R&D Systems), according to manufacturer’s instructions. IL-1β and IL-4 analysis of 24-h co-culture supernatants was performed using the MSD Mouse V-PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics, Rockville, MD). Briefly, biological triplicates of cell-free supernatants were run in duplicate and analyzed on a QuickPlex SQ120 analyzer using MSD Discovery Workbench 4.0 software (Meso Scale Diagnostics, Rockville, MD). Assay sensitivity using this platform is 0.04 pg/ml.
Whole cell lysates from differentiated muscle cultures were prepared using the NP40 Lysis buffer (Invitrogen) containing anti-proteases (Mini-Complete, Roche, Indianapolis, IN). Protein concentration was determined by the bicinchoninic acid assay (BCA) (Thermo Fisher, Waltham, MA). To assay for IL-1β, 240-ng protein per well was assayed in biological triplicates using the Mouse IL-1β Quantikine ELISA kit (R&D Systems).

Serum IL-1β activity was assessed in serum samples using a mouse IL-1β Quantikine ELISA Kit (MLB00C, R&D Systems) according to the manufacturer’s instructions.

Fresh testis samples were homogenized to obtain the supernatant fraction. The testosterone levels and IL-1β and MMP-8 levels were quantified following the manufacturer’s protocols. Testosterone was measured using a testosterone (mouse/rat) ELISA Kit from Biovision (#K7418-100). The IL-1β levels were evaluated using a mouse IL-1β Quantikine ELISA Kit from R&D Systems (#MLB00C, USA). The MMP-8 levels were detected using an MMP-8 (Mouse) ELISA Kit from Abnova (#KA5109). The caspase 1 activity in the testes and cell extracts was measured using a Caspase 1 Colorimetric Assay Kit (#K111, BioVision, Mountain View, USA). The NADPH oxidase activity in the testes and cell extracts was determined using a commercial kit (Nanjing Institute of Jiancheng Bioengineering).

Blood samples were obtained by intracardiac puncture in EDTA tubes and centrifuged for 20 min at 2000×g to collect plasma. Peritoneal fluid was collected by washing the peritoneal cavity with 2 ml of DPBS, and then centrifuged for 3 min at 1000×g to remove peritoneal cells. Samples were analyzed using the mouse IL-1β Quantikine ELISA kit or IL-6 Duoset ELISA kit (R&D Systems).