Measuring IFN-γ Production in Brucella Infection

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)].