Measurement of CD8+ T cell cytotoxicity

1. B16F10 cells were seeded in 96-well plates at a density of 1×10⁵ cells per well and incubated for 24 h in RPMI 1640 medium containing 10% fetal bovine serum (FBS). 

2. The medium was then removed and B16F10 cells were incubated with activated pmel-1 CD8⁺ T cells, Ava-pretreated pmel-1 CD8⁺ T cells, and pmel-1 CD8⁺ T-Tre/ 

BCN-Lipo-Ava cells at a density of 1×10⁶ cells per well in 200 μl phenol-free RPMI 1640 medium containing 2% FBS for another 4 h, 12 h or 24 h. Additionally, 200 μl phenol-free RPMI 1640 medium containing 2% FBS without T cells were added into the 96-well plate with B16F10 cells as controls.

3. Then, the mixed cells were centrifugated at 400 g for 5 min using a plate centrifuge. The supernatant was collected for measuring the release of endogenous lactate dehydrogenase (LDH) from B16F10 cells using the LDH Cytotoxicity Assay Kit (Beyotime Biotechnology) according to the instruction of the manufacturer.

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