Corticosterone elisa kit

1 ml cardiac blood was sampled in the morning and centrifuged at 3000 r/min under 4 degrees Celsius for serum isolation. The supernatant was kept at −80 degrees before measuring with Corticosterone ELISA kit (Abcam). The procedure was performed following the kit brochure, and the corticosterone concentration was calculated from the standard curve.

Blood samples of mice were collected by heart puncture under 20% urethane anesthesia on the day of sacrifice between 9 am to 11 am. Serum was taken from blood samples centrifuged at 1,500 × g for 20 min and stored at −80 °C. Serum corticosterone levels were measured using Corticosterone ELISA kit (Abcam LLC, Cambridge, UK) in accordance with manufacturer’s instructions. The serum concentrations of TNFα were measured using a commercial ELISA kit (Biolegend); a 100 μl serum sample and assay diluent were placed in each well of a 96-well plate coated with a monoclonal mouse IgG against TNFα. The mixtures were incubated for 2 h at 4 °C, followed by aspiration and three washes with washing buffer. Subsequently, 200 μl conjugate solution was placed into each well and incubated for 2 h at room temperature. After washing each well four times with washing buffer, 200 μl of substrate solution prepared with equal amounts of stabilized hydrogen peroxide and tetramethylbenzidine was added for a 20 min reaction in the dark. The reaction was quenched by adding 50 μl stop solution (2NH₂SO₄)64 (link).

C57BL/6 mice (National Cancer Institute, Charles River Laboratories; strain code 556) and their transgenic derivatives were maintained in the Hospital for Special Surgery and Weill Cornell Barrier Animal Facility in full compliance with the protocol approved by the Institutional Animal Care and Use Committee. Homozygous WT (wt/wtGRIP1^fl/fl) and GRIP1 KO (LysM-CreGRIP1^fl/fl) mice were generated previously^{31 (link)}.
GRIP1 KO and WT mice were injected intraperitoneally with 5 mg kg⁻¹ of LPS (Sigma) and monitored for 36 h. Mice were weighed at the time of injection and 24 h later.
For sorting of peritoneal macrophages, animals were euthanized via CO2 inhalation after 12 h of 5 mg kg⁻¹ LPS i.p. administration and peritoneal gavages with PBS performed. Antibody staining for flow cytometry included: CD45-PerCP-C5.5 (anti-mouse CD45, clone 30-F11), Cd11b-PE (anti-mouse/human CD11b, clone M1/70), CD3-FITC (anti-mouse CD3e, clone 145-2C11), CD11c-FITC (anti-mouse CD11c, clone N418), Gr1-FITC (anti-mouse Ly-6G/Ly-6C (Gr1), clone RB6-8C5), B220-FITC (anti-mouse/human CD45R/B220) (Biolegend; 1:100 dilution each).
Serum GC levels were determined using the Corticosterone ELISA kit (Abcam, ab108821) and as per manufacturer’s directions. Serum was collected from the blood of euthanized mice12 h after 5 mg kg⁻¹ LPS i.p. injection.