Mouse elisa kit

RAW264.7, ΔTLR1, ΔTLR4, ΔTLR2, ΔTLR6, ΔMyd88, and ΔMyd88/TRIF mouse macrophage cells were seeded (~1 × 10⁶ cells per well) in a 12-well-culture plate and left for 2 h at 37°C for adherence. After adherence, cells were treated with various concentrations of recombinant RipA protein (0.5, 1, and 2 μg/ml) or lipopolysaccharide (LPS) (1 μg/ml; positive control) (Sigma, USA). The protein treatment dose was pre-standardized for optimal release of cytokines and other cellular markers. Heat-inactivated (HI) RipA protein-treated cells were used as negative controls for cytokine production. After 24 and 48 h of treatment, supernatants were collected and stored at −80°C until used. The estimation of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, and anti-inflammatory cytokine, IL-10, was performed using mouse ELISA kit (BD Biosciences, San Jose CA, USA) following the manufacturer's instructions.

At the endpoint of prophylactic and combination cancer immunotherapy, the tumour sites and spleen were excised and lysed with a T-PER tissue protein extraction reagent (Thermo Fisher Scientific), and the amounts of cytokines in tumour sites were quantified using mouse ELISA kit (BD Biosciences) according to the manufacturer’s instructions.

Total IgE cytokine levels in sera and detection of IFN-γ, IL-5, and IL-4 in cultured splenocytes were performed using a mouse ELISA kit purchased from BD Biosciences (USA). It was measured and quantified in accordance with the protocol provided by BD Biosciences.

RAW 264.7 cells were seeded at a density of 1 × 10⁶ cells/well in a 6-well flat-bottom plate and concentrations of bacterial strains (5 mg/mL) were added for 24 h. Cell-free supernatants were harvested to measure the production of TNF-α, IL-6, and IL-1β using a mouse ELISA kit (BD Biosciences, East Rutherford, NJ, USA). Absorbance was measured at 450 nm using a microplate reader (BMG Lactobacillustech, Ortenberg, Germany).

Effect of bacterial strains on cytokine production by primary mouse splenocytes was assessed. Cells were seeded at a density of 1 × 10⁶ cells/well in a 6-well flat-bottom plate with or without 10 μg/mL of Concanavalin A (ConA) and incubated for 72 h. Then, cell-free supernatants were harvested to measure the production of TNF-α and IFN-γ using a mouse ELISA kit (BD Biosciences). Absorbance was measured at 450 nm using a microplate reader.