Ifn γ elispot assay kit

IFN-γ production in tumor microenvironment was detected with IFN-γ ELISPOT assay kit (BD Biosciences, 551881). Tumor tissues were harvested at 8-12 days after transplantation. Tumor tissues were digested with DNase I (Calbiochem) and Liberase TL (Roche) at 37 °C for at least 1 h. The cell suspension was passed through a 70 μm filter (ThermoFisher Scientific) and pelleted by centrifugation at 1500 rpm for 5 min. The cell pellets were resuspended by red blood cell lysate (Solarbio) on ice for 15 minutes to remove red blood cells. Then cell suspension was centrifugated at 1500 rpm for 5 min and washed by PBS twice. 2 × 10⁶ cells were seeded per well in a pre-coated with purified anti-mouse IFNγ antibody (1:200) plate (EMD Millipore) and incubated incubator with 5% CO₂ at 37 °C for 16-24 hours. The next day, cell supernatant was removed, and the plate was washed by 1640 medium containing 10% FBS once and then blocked with 1640 medium to block nonspecific sites of antibody. Then plate was treated with biotinylated anti-mouse IFNγ antibody (BD, #511818KZ) for 2 h and streptavidin-HRP for 1 h respectively. The reaction stopped by adding final substrate solution. The plate was scanned using CTL ImmunoSpot® S6 Analyzers (CTL and CTL Analyzers, LLC) and red dots were counted after washed with ddH₂O and completely dry.

The IFN-γ ELISpot assay kit (BD Biosciences, San Jose, CA, USA) was used to determine Ag85B and ESAT-6 specific responses of cells isolated from spleens, lungs and cervical lymph nodes using a previously described protocol [23 (link)]. Aliquots (2 × 10⁵) of cells from the different tissues were cultured in duplicate with either Ag85B (5 μg/mL), ESAT-6 (5 μg/mL), or Concanavalin A (5 μg/mL) for 48 h at 37 °C and 5% CO₂. Enumeration of spots representing individual cells producing IFN-γ was conducted by Zellnet Consulting Inc., Fort Lee, NJ, USA) using KS-ELISPOT automatic system (Carl Zeiss Inc., Thornwood, NY, USA). Responses were considered positive only when they were above 10 Spot forming Cells (SFC)/2 × 10⁵ input cells and at least twice the number obtained in cells cultured with medium alone.

dLN cells were resuspended in RPMI 1640 medium supplemented with 10% FBS, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. A total of 3 × 10⁵ dLN cells was used for the assay. A20 cells were irradiated with a single dose of 60 Gy. The ratio of A20 to immune cells was 1:4. After 48 hours of incubation, IFN-γ production was determined with an IFN-γ ELISPOT Assay Kit (BD Biosciences) according to the manufacturer’s protocol. The visualized cytokine spots were enumerated with an ImmunoSpot S6 Analyzer (Cellular Technology Limited).