Cd8a fitc

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.

Methotrexate (MTX, 2.5 mg per tablet) was purchased from XINYI Medical Limited Company in Shanghai, China. MTX injection (0.1 g per ampoule) was purchased from Pfizer, Inc. (NY, USA). Paroxetine, with chemical name (3S,4R)-3-((Benzo[d][1,3]dioxol- 5-yloxy) methyl)-4-(4- fluorophenyl) piperidine hydrochloride was purchased from Ark Pharm, Inc. CCII was obtained from Chondrex Corporation. Enzyme-linked immune sorbent assay (ELISA) kits for IL-1β, TNF-α and CX3CL1 were the products from Raybiotech, Inc. (GA, USA). Rabbit anti-rat GRK2, rabbit anti-rat CD3 and rabbit anti-rat β-actin were purchased from Santa Cruz Biotechnology, Inc. (CA, USA), rabbit anti-rat pERK (Thr202/Tyr204) and rabbit anti-rat ERK were ordered from Cell Signaling Technology, Inc. (China). CX3CL1 cytokine was obtained from Peprotech Inc. (Jiangsu, China). PE-CD3, FITC-CD4, PE-CD25, PE-CD8a, FITC-CD8a, PE-CD44 and Regulatory T cell detection kit were procured from eBioscience, Inc. (CA, USA). PE-CD62L was purchased from BioLegend, Inc. (CA, USA). FITC-CD11b is obtained from BD Biosciences (NJ, USA). Anti-TCRγ/δ magnetic beads were the product of Miltenyi Biotec GmbH (Germany).