Ifn γ elispot kit

All ELISpot-specific reagents are part of the IFNγ-ELISpot kit from BD Biosciences (Cat# 551083). ELISpot plates were coated overnight at 4°C with anti-IFNγ antibody. Plates were washed and blocked with DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 mM HEPES for 2 hr at RT. Spleens were harvested from mice and mashed through a 70 μm filter with a 1 mL syringe plunger to generate a single-cell suspension. Red blood cells were lysed with 500 μL of ACK Lysing Buffer (Gibco) on ice for 5 min, and splenocytes were washed three times with chilled PBS. For IFNγ-ELISpot assays using peptide restimulation, 1 × 10⁶ splenocytes were assayed per well in the presence or absence of 10 µg of peptide. As a positive control, splenocytes were incubated with a mixture of 100 ng/mL PMA (Sigma-Aldrich) and 1 μg/mL ionomycin (Sigma-Aldrich). Following an overnight incubation at 37°C and 5% CO₂, plates were developed using the BD mouse IFNγ-ELISpot kit, following manufacturer’s protocol.

Two weeks after the final immunization, mice were euthanized via decapitation. Single-cell suspensions of splenocytes cells were obtained by gentle mechanical disaggregation through sterile 100-um nylon cell strainers (BD Falcon). For the lymphocyte proliferation assay, cells were added to 96-well flat-bottomed tissue culture plates at 5 × 10⁵cells/well and stimulated with VP1_237-249 protein (10 μg/ml). Plates were cultured at 37 °C in a humidified incubator with 5% CO₂. Three days later, cell proliferation was performed using an ELISA BrdU-Kits (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
IFN-γ producing cell frequency in spleens was assessed using IFN-γ ELISPOT kit (BD Pharmingen). Briefly, cells were plated at 1 × 10⁶cells/well and stimulated with VP1_237-249 protein (10 μg/ml) for 48 h at 37 °C with 5% CO₂. After sequential incubation with biotinylated detection antibody, streptavidin-HRP and AP-colorimetric substrate, color was developed and spot-forming cells were enumerated with an ImmunoSpot Series 3 Analyzer (Cellular Technology).

The bacillary load (BL) and specific IFN-γ responses measured were the parameters chosen to evaluate the infection. Both parameters were measured in lung, spleen and lymph nodes, which were extracted and mechanically disrupted in order to obtain tissue homogenates. Lymph nodes samples could occasionally not be harvested at the earliest timepoints as they were too small to be readily recognised, therefore no results were recorded in such cases.
The BL was determined by culturing the samples on Middlebrook 7H11 agar plates (Bennex Ltd, Shannon, Ireland) at 37°C for 21 days. Visible colony forming units (CFU) were counted four weeks later, with data being recorded as the log of the total number of CFUs recovered per organ.
Specific cellular IFN-γ responses against the antigens ESAT-6 (Lionex Diagnostics & Therapeutics, Braunschweig, Germany), which was used at a final concentration of 5 µg/mL, and PPD (Statens Serum Institute, Kobenhavn, Denmark), which was used at a final concentration of 10 µg/mL, were measured using an IFN-γ ELISPOT kit (BD Bioscience, San Diego, CA) after plating 250,000 cells from the tissue homogenates. The ELISPOT assay was further developed and read according to the manufacturer's instructions. The results were expressed as IFN-γ secreting cells as Spot Forming Units (SFU) per 250,000 cells.