Elisa max standard kit

The supernatants of co-cultures of tumor and CD8+ T-cells were collected after overnight incubation and stored at -80°C. IFN-γ levels from the co-cultures were measured by ELISA Max Standard kit (BioLegend) in 96-well microtiter plates according to the manufacturer’s instructions. Results are expressed as pg/mL.

U937 cells (1 × 10⁵ cells/well) were pre-incubated in a 48-well tissue culture plate with 100 nM PMA for 48 h. After exchanging the medium with pre-incubated fresh RPMI medium 1 h prior to infection, cells were infected with bacterial strains (F. novicida transposon mutant strains or deletion mutant strains) at an MOI of 1 or stimulated with 100 ng/ml of LPS derived from E. coli (O127:B8) or 10 ng/ml of 2′3′-cGAMP (InvivoGen). Heat-inactivation of each strain was performed by incubating the bacterial suspension in a heat block at 90°C for 5 min. In the case of STING inhibition, a STING-specific inhibitor, H-151 was used (Haag et al., 2018 (link)). U937 cells were treated with H-151 (final concentration 0.5 μM) or the same volume of DMSO 2 h prior to infection. After incubation for the indicated time durations in figure legends, concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the supernatants were measured using ELISA MAX Standard Kit (Biolegend, CA, USA) according to the manufacturer’s instructions. Interferon (IFN)-β in the supernatants were measured using VeriKine Human Interferon Beta ELISA Kit (PBL Assay Science, Piscataway, NJ, USA).