E el m0037

Serum concentrations and cell culture supernatant medium level of IL-1β were measured by the corresponding ELISA kits (Elabscience, E-EL-M0037) according to the manufacturer's instructions.

The levels of the pro-inflammatory cytokines IL-1β and IL18 were measured using commercially available kits according to the manufacturer’s instructions (Elabscience, United States, catalog number: E-EL-M0037 for IL-1β and E-EL-M0730 for IL-18). In brief, standards and supernatants of homogenates were added to the 96-well plates coated with the specific murine monoclonal antibodies raised against IL-1β or IL-18. Plates were then incubated for 90 min at 37°C followed by decantation of the unbound antibodies. Secondary biotin-labeled specific antibodies for IL-1β and IL18 were added and incubated for 60 min at 37°C. Following this incubation, the plates were washed with the supplied washing buffer before applying the HRP conjugate for 30 min at 37°C. Finally, the substrate reagent was added, and the plates were incubated for 15 min at 37°C followed by the addition of the stop reagent. The absorbance of the developed color was measured at 450 nm. The concentration of inflammatory cytokine in the brain tissues was extrapolated from the standard curve, expressed as pg/ml.

The effects of the extracts on levels of TNF-α, IL1 β and IL10 were determined and quantified using the mouse ELISA kits with catalogue numbers E-EL-M0049, E-EL-M0037 and E-EL-M0046, (Elabscience, Biocom Africa, Johannesburg South Africa)), respectively, following the same protocol as above using the instructor manual on the cell culture medium.