Mouse t cell activation and expansion kit

CD8 and CD4 T cells were isolated from mouse spleen (T-cell isolation kits were ordered from Miltenyi Biotec). After 24-h activation by CD3ε and CD28 beads (Mouse T cell Activation and Expansion Kit, Miltenyi Biotec #130-093-627), the cell suspension was diluted to 2 × 10⁵ T cells/mL (50% CD4 and 50% CD8 T cells). In all, 100 μL cell suspension (2 × 10⁴ T cells) was added into each well of a 96-well plate. The T cells were treated by PBS or 5 μL (0.01 mg/mL) PL1-OX40 (WT) or PL1-OX40 (ψ) for 12 h. Then, CD3ε and CD28 beads were removed. Lastly, the T cells were stained with OX40-FITC antibody and then detected by flow cytometry. Similarly, T-cell proliferation and activation were performed, respectively. The T cells were labeled by CellTrace™ CFSE (CellTrace™ Cell Proliferation Kits, CellTrace™ CFSE, C34570) and were resuspended in the medium containing CD3 beads and 5 μg/mL anti-OX40 Ab. After 60 h, the cell number was analyzed by a flow cytometer with a 488 nm excitation source. Additionally, intracellular IFN-γ and TNF-α were quantified by ELISA.

Temoporfin (Foscan^®, 5,10,15,20-Tetrakis(3-hydroxyphenyl)chlorin (mTHPC)) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Rat anti-mouse CD8 antibody was purchased from BD Bioscience (San Jose, CA, USA). Recombinant interleukin-2 (IL-2) was purchased from PeproTech (Cranbury, NJ, USA). RPMI 1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Welgene Inc. (Gyeongsangbuk-do, Republic of Korea). TUNEL assays kit was purchased from R&D systems (Minneapolis, MN, USA). Mouse T cell activation and expansion kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Mouse CD8 T cell enrichment columns were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Cell counting kit-8 (CCK-8) and WST-1 colorimetric assays were purchased from Vitascientific (Beltsville, MD, USA) and Roche Diagnostics GmbH (Mannheim, Germany), respectively.