Elisa max standard set mouse tnf α

MH‐S cells were plated in 12‐well plates at a density of 7.5 × 10⁵ cells per well. Cells were treated with 50 µg mL⁻¹ of SLAP‐S25, GNGs, and 10 µm dexamethasone for 30 min before the final concentration of LPS (1 µg mL⁻¹) was added to cell cultures. After 4 h of incubation, the supernatants were obtained for TNF‐α and IL‐6 production analysis using ELISA MAX Standard Set Mouse TNF‐α and ELISA MAX Standard Set Mouse IL‐6 (Biolegend, US).
The expression of TNF‐α and IL‐6 relative to β‐actin was detected by qRT‐PCR tests with the PowerUp SYBR Green Kit (Applied Biosystems). Thermal cycling was performed using a two‐step PCR amplification standard procedure at 95 °C for 30 s and 40 cycles of 60 °C for 30 s and 72 °C for 30 s. The qRT‐PCR test was performed using the ABI Quantstudio 7 detection system (Applied Biosystems). The fold changes of gene expression were determined using the 2^−ΔΔCt method.

Serum samples were obtained from normal control and SSHTN mice. TNF-α concentration was measured using ELISA MAX Standard Set Mouse TNF-α (Biolegend, United States) according to the manufacturer’s protocols. 96-well ELISA plates (Biolegend) were precoated with monoclonal hamster antibody in carbonate buffer and incubated overnight at 4°C. To block non-specific binding and reduce background, the plates were incubated for 1 h at room temperature with the addition of 1% bovine serum albumin (BSA; Sigma-Aldrich) in PBS. Diluted mouse TNF-α standard and serum samples were added to the wells and incubated for 2 h at room temperature. Biotinylated goat polyclonal anti-mouse TNF-α detection antibody was added to the wells with 1 h incubation at room temperature. The bound IgG was detected by incubation with HRP-conjugated avidin, followed by colorimetric detection with TMB substrate solution (Biolegend). The reaction was stopped with 2 N H₂SO₄ after 30 min, and the absorbance was measured at 450 nm using a microplate reader (Remote Sunrise, Tecan, Japan). Absorbance value at 595 nm was used as a reference.