Mouse il 1 beta il 1f2 quantikine elisa kit

Immune cell fraction was prepared from mouse liver as described above. Fractionated immune cells were lysed and protein isolated using an acid-guanidinium-phenol based reagent TRIzol (ThermoFisher, GE17-0891-01) according to manufacturer’s instructions. Protein was concentrated using Protein Concentrator polyethersulfone (PES), 10K molecular weight cutoff (MWCO) (ThermoFisher, 88503). Protein was quantified using colorimetric Pierce BCA Protein Assay Kit (ThermoFisher 23225). Protein was run on 12.5% gel and transferred to polyvinylidene difluoride (PVDF) membrane. Membranes were blocked using 5% bovine serum albumin (BSA) for 1 hour. Primary antibodies were diluted in Tris-buffered saline (TBS) (0.2% Tween-20) buffer and incubated at 4°C overnight. Primary antibodies used: caspase-11 (ThermoFisher, 14-9935-82), GSDMD (Abcam, ab209845), and β-Actin (Sigma-Aldrich, ab6276). Secondary antibodies were diluted in TBS (0.2% Tween-20, 0.01% SDS) and incubated for 30 minutes to 1 hour at RT. Secondary antibody used: IRDye 680RD (LI-COR), IRDye 800CW (LI-COR). Membranes were scanned using LI-COR Odyssey Crx (Li-Cor Biosciences, Lincoln, NE). Image processing was performed using Image Studio Analysis (LI-COR). Liver IL-1β levels were assessed using the Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (R&D Systems, MLB003) following the manufacturer’s instruction.

Spinal cord tissue cytokines were assayed using ELISA kits for IL-1β (Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit, MLB00C), IL-6 (Mouse IL-6 Quantikine ELISA Kit, M6000B), ICAM1 (Mouse ICAM-1/CD54 Quantikine ELISA Kit, MIC100) and IL-10 Mouse IL-10 Quantikine ELISA Kit, M1000B), which were all from R&D Systems. The assays were performed according to the manufacturer’s instructions.

IL-1β released into cell culture supernatants after various treatments was quantitated using the Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (R&D- Cat No. MLB00C) according to the manufacturer’s protocol. The standard curve was generated using the kit’s standards.

Microglial TNF-α and IL-1β secretion was measured in cell supernatants using the Mouse TNF-alpha Quantikine ELISA Kit (SMTA00B, R&D Systems, Minneapolis, MN, USA) and Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (SMLB00C, R&D Systems) according to the manufacturer’s protocols.

Tumor homogenates were generated in PBS that was supplemented with a cocktail of protease inhibitors (Roche) using a pestle inside an Eppendorf. The conditioned media of cultured splenocytes and MDSCs from in vitro cultures were also collected. The homogenates and cell culture supernatants were centrifuged and were assayed for mouse IL-1β using the Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (R&D Systems, MLB00C), according to the manufacturer’s instructions.

Using 100 μl of plasma, plasma levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured using commercially available kits, TNF-α kit (Mouse TNF-alpha Quantikine ELISA Kit, R&D Systems, MN, United States) and IL-1β kit (Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit, R&D Systems), respectively.