Spleens from mice adoptively transferred with DCs were aseptically dissected after euthanasia and spleen cells were separated using a 40 μm cell strainer (Corning Cell Strainer, Corning Incorporated, Durham, NC, United States). A cell suspension was prepared and transferred to 96-well plates at 105 cells/well followed by stimulation with concanavalin A (Con A-0.5 μg/mL final concentration; Sigma-Aldrich, St Louis, MO, United States) or heat-killed R. rickettsii (HKRr−2 × 105 bacteria/well) and incubated at 37°C under 5% CO2. After 48 h, 25 μL of 0.01% resazurin (prepared in complete medium) were added to culture cells and after additional 24 h, the culture absorbance at 570 and 600 nm was determined and used to indirectly evaluate cell proliferation as previously described (31 (link), 36 (link), 49 (link)).
In another set of experiments, spleen cells were distributed into 24-well plates at 2.5 × 106 cells/well followed by stimulation with 5 × 106 cells of HKRr/well. After 72 h of incubation at 37°C under 5% CO2, the concentration of IFN-γ and IL-4 was evaluated in cell-free supernatants according to manufacturer's instructions (BD OptEIA™ ELISA Set-BD Biosciences).
In another set of experiments, spleen cells were distributed into 24-well plates at 2.5 × 106 cells/well followed by stimulation with 5 × 106 cells of HKRr/well. After 72 h of incubation at 37°C under 5% CO2, the concentration of IFN-γ and IL-4 was evaluated in cell-free supernatants according to manufacturer's instructions (BD OptEIA™ ELISA Set-BD Biosciences).
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