Annexin 5 and pi

Human leukemic cell lines were cultured in RMPI-1640 medium (Life Technologies) with 10% fetal bovine serum (Life Technologies). For differentiation analyses, 1 µM ATRA and/or 10 µM TCP (in DMSO) was added to the cultures. Forty-eight hours later, cells were labeled with CD11b-PE (BD Biosciences) or CD34-FITC (BD Biosciences) antibodies and PI (Biolegend) and analyzed by Accuri C6 flow cytometer (BD Biosciences). Apoptotic cells were analyzed by labeling with Annexin V and PI (BioLegend) 4 d after shRNA virus infection.

Cytotoxicity was determined by a calcein-AM release assay as previously described.^{8 (link)} Briefly, the parental and A2⁺CLDN6⁺ A2780 were labeled with 5 μM calcein-AM (Sigma-Aldrich) and mixed with CD8-TCR- or mock-transduced T cells for 4 h at E/T ratio, 50, 25, or 12.5. Fluorescence release in cell-free supernatants was measured by the Synergy H1 microplate reader (BioTek) with excitation 485 and emission 528. To induce the maximum release, target cells were treated with 2% Triton-X, whereas the spontaneous release was determined from supernatants of target cell alone. Cytotoxicity was calculated using the following formula: % cytotoxicity = 100 × [(experimental release – spontaneous release)/(maximum release – spontaneous release)]. To evaluate the induction of apoptotic cell death of cancer cells by CD8-TCR-transduced T cells, the parental and A2⁺CLDN6⁺ A2780 (5 × 10⁵ cells) were cocultured with T cells (1 × 10⁶ cells) for 18 h. Cells were harvested with 0.25% trypsin–EDTA solution and stained with anti-CD3 antibody (clone UCHT1, BioLegend) followed by annexin V and PI according to the manufacturer’s instruction (BioLegend). Percentages of Annexin V⁺ and PI⁺ cells were analyzed for CD3⁻ cells.