PBMCs were seeded in 96-well round-bottom plates (5 to 8 × 105 cells per well) and infected with the different Salmonella strains from frozen midlog phase stocks, at the indicated MOI. Upon 80-min incubation at 37 °C, 100 μg/mL gentamicin (Lonza) was added to kill extracellular bacteria. A total of 200 μg/mL of gentamicin was required for the experiments carried out in Malawi using ST313 strains from lineages 1 and 2.
At 180 min postinfection, 5 ng/mL brefeldin A (BioLegend) solution was added to every well in order to achieve accumulation of intracellular cytokines. Samples were incubated overnight at 37 °C for no more than 15 h.
For MR1 blocking experiments, infection was performed in the presence of 30 μg/mL MR1 blocking antibody 26.5 (42 (link)) or mouse IgG2a isotype control (ATCC). The MAIT agonists 5-A-RU was synthesized as described in ref. 71 (link) and 1 μg/mL was combined to 50 μM MG (Sigma).
At 180 min postinfection, 5 ng/mL brefeldin A (BioLegend) solution was added to every well in order to achieve accumulation of intracellular cytokines. Samples were incubated overnight at 37 °C for no more than 15 h.
For MR1 blocking experiments, infection was performed in the presence of 30 μg/mL MR1 blocking antibody 26.5 (42 (link)) or mouse IgG2a isotype control (ATCC). The MAIT agonists 5-A-RU was synthesized as described in ref. 71 (link) and 1 μg/mL was combined to 50 μM MG (Sigma).
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