Mouse ifn γ

AB1 cells were cultured in triplicates (5 × 10³ cells/well) in 96 well plates. Mouse IFN-γ (Biolegend, San Diego, CA) was added in serial concentrations (0.3, 0.9, 1.2, 5 and 10 U/ml) in 150 μl of medium. After 3 days, cell proliferation assay WST reagent (Roche) was added for 3 h to the culture medium according to the manufacturer’s instructions. The absorbance was measured at 480 nm.

After euthanasia using a CO₂ chamber at 3 weeks after immunization, the serum in whole blood by cardiac puncture was collected for mouse IFN-γ (Cat# 430807, BioLegend, San Diego, CA, USA), IL-17 (Cat# 432507, BioLegend), TNF-α (Cat# 430907, BioLegend), and IL-1β (Cat# BMS6002, Invitrogen) analyses. ELISA kits were analyzed from accordance with the directions provided by the manufacturer. The samples were then analyzed with an ELISA microtiter plate auto reader at 450 nm (Molecular Devices, San Jose, CA, USA).

Blood samples of five mice from each group were collected 4 and 6 weeks p.i.; IFN-γ was measured using an ELISA kit (Mouse IFN-γ Biolegend-USA). Additionally, 6 weeks p.i., five mice from each group were sacrificed, their spleens were separated aseptically, and their splenocytes were grown and infected by RB51 and the RB51 recombinant containing mLLO-BAX-SMAC to determine IFN-γ production [33 (link)]. B. abortus RB51 and the RB51 recombinant containing mLLO-BAX-SMAC were suspended in PBS, washed thrice, and resuspended in PBS with a concentration of 10⁸ CFU. For heat inactivation, the bacterial suspension was incubated in a 65 °C water bath for 60 min. To confirm complete inactivation, aliquots of the resulting bacterial suspensions were spread onto TSA plates and incubated at 37 °C for five days [34 (link)]. Splenocytes from the inoculated mice were obtained as previously described and grown in 10⁸ heat-inactivated B. abortus RB51 and RB51 recombinant containing mLLO-BAX-SMAC per well in the related groups. The cells were grown for 5 days, and their supernatants were collected. The sera and collected supernatants were tested for IFN-γ, with recombinant Mouse IFN-γ as the standard. The assays were performed in triplicate [30 (link)].

Mouse IFN-γ (cat. 430805), TNF-α (cat. 430905), IL-12 (cat. 433605), IL-4 (cat. 431105), and IL-6 (cat. 431305) ELISA kits were purchased from BioLegend (San Diego, CA, USA), and ELISA was performed according to the manufacturer’s protocols.

BMDM were prepared by collecting bone marrow from the femurs of mice and then plating the cells in Petri dishes in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS: Invitrogen Life Technologies), and 10% macrophage colony-stimulating factor (M-CSF)-conditioned medium. The latter was collected from the L929 cell line. After incubation at 37°C in 5% CO₂ for 6 days, FCS-BMDM were harvested and added to 6-, 24-, or 96-well plates at the indicated density, and incubated overnight before infection. When applicable, BMDM were treated with 100 units/ml of mouse IFN-γ (#575306; Biolegend, San Diego, CA).