Annexin 5 alexa647

Detection of apoptosis was performed with the aid of Annexin-V TM 633 (Life Technology, USA) combined with SYTOX^® Green dye (Life Technology, USA) staining. HCT-116 cells were treated with CL-43 at a concentration of 250 nM (20 h), alone or in combination with anti-cancer drugs etoposide (200 μM) or cisplatin (50 μM) 20 h later, cells were collected, washed in cold PBS, resuspended in the binding buffer provided by the manufacturer, and stained with Annexin-V-Alexa647 and SYTOX^® Green (Life Technology, USA) according to manufacturer’s recommendations. The cell cycle was then measured with the aid of the CytoFlex Flow FACS (Beckman Coulter, USA) using laser with λ = 488 nm and analyzed with ModFit LT (Verity Software House Inc, USA) software.

Control cells and cells co-cultivated with THP1 or monocytes treated with etoposide or chloroquine were used in the flow cytometry assay. Cells were seeded into 12-well plates at a concentration of 10⁵ cells/well. The next day, simultaneously with the medium changing, 5 × 10³ THP1 cells were added. After 20 h of co-cultivation, chloroquine (200 μm) or etoposide (25 μm) were added without changing the medium. Further plates were cultivated for the next 36 h in the incubator and then cells were prepared for flow cytometry as described in our previous works [21 (link)] The detection of apoptosis was carried out with the aid of Annexin V Alexa 647 (Life Technology, Carlsbad, CA, USA) combined with Propidium Iodide staining. All procedures were performed according to the manufacturer's protocol.

For Phospho-flow, 5 × 10⁵ 1G4-CD8 cells were stimulated with 200 nM tetramers in RPMI at 37°C. Samples were fixed immediately in fixation buffer (BD Bioscience) at 37°C for 10 min, washed with PBS, 1% BSA, 0.01% azide before permeabilization with 10% saponin on ice for 30 min (Perm/wash buffer 1, BD Bioscience). Antibody staining was for 1 h at RT in 10% saponin buffer. Flow cytometry was performed on a FACSCalibur (Becton Dickinson), and the experiment was analysed using FlowJo (Tree Star, Inc.). Statistical analysis was performed in GraphPad Prism. To detect apoptosis, 2 × 10⁵ 1G4-CD8 cells were stimulated with 20 nm pMHC tetramers or 2 μg/ml camptothecin for 24 h at 37°C in RPMI with 10% FCS. After incubation, cells were stained with FAM- or far-red-FLICA (Immunochemistry Technologies). For plate-bound stimulations, biotinylated 6V monomers were immobilized at the indicated concentrations on Streptavidin-coated 96-well plates (Thermo Scientific) at 4°C overnight in PBS. Plates were washed twice with PBS, and 10⁵ 1G4-CD8 cells/well were incubated ON at 37°C in RPMI with 10% FCS. Cells were harvested, washed in PBS and stained with Annexin-V-Alexa647 (Life Technologies) in 100 mM HEPES, 140 mM NaCl, 25 mM CaCl₂, pH 7.4. Flow data were analysed as described above.