Apc tcrb

After 10 days of incubation, each co-culture well was resuspended at 2 × 10⁶ cells per mL in 400 μL T cell medium and split into two aliquots (200 μL each). Cells were washed twice in FACS buffer and stained with the following antibodies (extracellular staining): FITC-CD8a (1:80)(Peprotech), Per55-I-A/I-E (1:200) (Peprotech), APC-TCRb (1:40) (Peprotech), at 4°C for 20 min. Cells were then washed twice in FACS buffer, fixed and stained (intracellular staining) for Granzyme B (PE-conjugated anti-GrB) (Peprotech), using the Cytofix/Cytoperm kit (BD), according to the manufacturer's instructions. PBMCs co-cultured with healthy organoids served as a negative control whereas PBMCs stimulated with 50 ng/mL PMA (Sigma-Aldrich) and 1 μg/mL Ionomycin (Sigma-Aldrich) served as a positive control; half of the contents of each co-culture well was incubated with PMA/Ionomycin solution for 5 h on ice.

In a parallel co-culture experiment, after 24 h incubation, co-cultured T cells were resuspended at 1 × 10⁶ cells per mL, washed twice in FACS buffer and stained with the following antibodies: PE-anti-CD137 (1:30) (Peprotech), FITC-CD8a (1:80) (Peprotech), Per55-I-A/I-E (1:200) (Peprotech), APC-TCRb (1:40) (Peprotech). PBMCs co-cultured with healthy organoids served as a negative control whereas PBMCs stimulated with 50 ng/mL PMA (Sigma-Aldrich) and 1 μg/mL Ionomycin (Sigma-Aldrich) served as a positive control.