Incucyte s3 platform

A375 GFP⁺ target cells were loaded with target peptide (custom ordered from Genescript) in LymphoONE supplemented with 1% human serum and 1× P/S as described above. Peptide-loaded A375 target cells were seeded in either 48-well (for “cycle” 1) or 96-well (for subsequent cycles) plates at 27,000/well or 9000/well, respectively. Twenty-four hours after peptide loading, T cells were mixed with target cells at 3:1 E:T ratio. Plates were placed in the IncuCyte S3 Platform (Sartorius) and imaged every 2 hours for 48 or 72 hours for cycle 1 and subsequent cycles, respectively. At the end of each cycle, cells in suspension were separated from adherent target cells and counted before added to the subsequent cycle of coculture with equivalently peptide-loaded target A375 cells and incubated for 72 hours. This process was repeated until T cells no longer killed peptide-loaded target cells (<10% loss of GFP signal observed in a given round). Data was reported as percent of control of the GFP signal relative to A375+ DMSO-only controls.

Fluorescein Isothiocyanate (FITC)-conjugated Annexin V (Biolegend, Cat #640906) and 7-amino-actinomycin D (7-AAD) kit (Biolegend, Cat #420403) was used to detect the apoptotic cells according to the manufacturer's instructions. After the treatment of 10 μM GSK126 for 48 h, the B16F10 cells were harvested with trypsin and washed twice with PBS. Cells were then resuspended in 1x binding buffer at a concentration of 1 × 10⁶ cells/mL. Next, 100 μL of the solution (1 × 10⁵ cells) was transfered to a 5 ml culture tube and 5 μL of FITC Annexin V and 5 μL 7-AAD were added into the solution. The mixture was incubated for 15 min at RT (25°C) in the dark. 200 μL of 1 x Binding Buffer was added to each tube and the apoptosis rate of the cells was analyzed by flow cytometry within 1 h.
Annexin V staining assay was detected by IncuCyte S3 platform (Sartorius, Göttingen, Germany). Cells were seeded in 96-well plates and treated with GSK126 and appropriate FITC Annexin (with binding buffer) on the next day. Four sets of phase contrast images from distinct regions within each well were taken at intervals of 6 h using a 10X objective with phase and green image channels.