Mouse il 18 elisa kit

Six-week-old male BALB/c mice were treated with PBS, SITO, or LS, as mentioned in the tumor model establishment section for 7 days prior to the sample collection. Mice were euthanized 3 h after the final administration, and the small intestines were removed and homogenized in the T-PER reagent containing 1% protease inhibitor cocktail. Homogenates were centrifuged at 12,000× g for 5 min, and the supernatants were collected and assayed with ELISA kits. The Mouse IL-12 p70 ELISA kit (M1270, R&D system, Minneapolis, MN, USA), mouse IL-18 ELISA kit (7625, MBL International, Nagoya, Japan), and mouse IFN-γ ELISA kit (MIF00, R&D system) were used to determine the IL-12, IL-18, and IFN-γ levels, respectively. The manufacturer’s instructions were followed, and the absorbance at 450 nm was measured using an ELISA plate reader (Bio-Tek).

For cytokine analysis by Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay (Bio-rad): 2 × 50 μl washes with sterile PBS were collected from the vaginal lumen using a pipette. Samples were centrifuged for 3 min at 13000*g to remove mucous and cells, and supernatants were added to 200 μl of ABC buffer. The assay was performed according to manufacturer instructions, and plates were read on a Luminex Bioplex 100 system (Biorad). For measurement of IL-18 or IFNβ, 2 × 50 μl washes with sterile PBS were collected from the vaginal lumen and centrifuged to remove mucous and cells. IL-18 was measured using the mouse IL-18 ELISA kit (MBL International) according to manufacturer's instructions at half-volumes, while IFNβ was measured using the LEGEND MAX Mouse IFNβ ELISA kit (Biolegend) according to manufacturer's instructions. For the measurement of IFNβ in tissue homogenates, vaginal tissues were collected in pre-weighed tubes and flash frozen on dry ice. NP40 lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP40 alternative) with protease inhibitor cocktail (Sigma Aldrich) was added to weighed tissue and bead-homogenized. Lysates were clarified by centrifugation and supernatants used for ELISA.