E el m0049

Several different kits for measuring parameters in mice, including mouse IL-1β (cat. No. E-EL-M0037), IL-6 (E-EL-M0044), and TNF-α (E-EL-M0049) were purchased from Elabscience (Houston, TX, USA). Serum cytokine levels were determined using Sandwich-ELISA kits in accordance with the product inserts and reference tables available at the Clinical Laboratory Sciences Department, Turabah University College, Taif University.

The levels of pro-inflammatory cytokines TNF alpha (TNF-α) and interferon-gamma (IFN-γ) were measured using commercially available kits (Elabscience, United States, catalog number: E-EL-M0049 for the TNF-α and E-EL-M0048 for IFN-γ) according to the manufacturer’s instructions. The experiments were conducted as described in our previous work^{43 (link)}. In brief, standards and supernatants of homogenates were added to the 96-well plates coated with the specific murine monoclonal antibodies raised against TNF-α or IFN-γ. Plates were then incubated for 90 min at 37 °C followed with gentle decantation and without any washing, the biotin-labeled specific antibodies for TNF-α and IFN-γ were added and incubated for 60 min at 37 °C. Following this incubation, wash with the kit washing buffer before applying the HRP conjugate for 30 min at 37 °C and after that. Finally, the substrate reagent was added and the plates were incubated for 15 min at 37 °C beforehand stopping the reaction and reading the absorbance of the developed color at 450 nm. The inflammatory cytokine contents in the brain tissues were expressed as pg/ml.