Annexin 5 binding buffer

Cells and supernatant were collected in fluorescence-activated cell sorting (FACS) tubes. For apoptosis, samples were washed with Annexin V binding buffer (BioLegend, 422201) and stained in Annexin V binding buffer with Annexin antibody (1:25; BioLegend, 640941, APC) and propidium iodide (1:1,000; Sigma, P4864) for 15 min (RT). For cell cycle analysis, samples were washed and fixed in 70% ethanol, and DNA was extracted with a solution of Na₂HPO₄ (0.2 M) and citric acid (0.1 M; pH 7.8) for 10 min (37 °C). Subsequently, samples were washed and incubated for 30 min (37 °C) with 40 μg ml^–1 propidium iodide (Sigma, P4864) and 100 μg ml^–1 RNase. Sample analysis was performed with a FACS Fortessa (BD Biosciences). Data were analyzed by FlowJo (TreeStar).

Cells were seeded in 12-well plates at subconfluent density (70%) and infected with each virus at an MOI of 5 without TPCK-treated trypsin. Twenty-four hours postinfection, cells were harvested using trypsin-EDTA and processed through staining for apoptosis markers. For this protocol, all spins were performed for 5 min at 4°C at 400 rcf and all solutions were used at 4°C unless mentioned otherwise. Cells were washed twice with 1 ml of PBS, resuspended in 100 μl of PBS with 1 μl of Zombie Red fixable viability dye (BioLegend), and incubated 30 min at 4°C in the dark. Then cells were washed twice with 1 ml of PBS–2% BSA and once with annexin V binding buffer (BioLegend) at room temperature (RT). Cells were incubated for 15 min at RT with 5 μl of annexin V (BioLegend) diluted in 100 μl of annexin V binding buffer. Then cells were washed once in annexin V buffer and once in PBS supplemented with calcium and magnesium (PBS-Ca-Mg). Cells were fixed in 200 μl of 2% formaldehyde diluted in PBS-Ca-Mg for 20 min at RT. Cells were washed twice in PBS-Ca-Mg and then resuspended in 200 μl of PBS-Ca-Mg. Samples were analyzed by flow cytometry using a Gallios 4 (Beckman Coulter) and results were analyzed with the software Kaluza Analysis 1.5a.

HL-1 cells were resuspended in Annexin V Binding Buffer (BioLegend). 100 µL of cell suspension were stained with 1 µL of FITC Annexin V (BioLegend) and 1 µL of 7-AAD Viability Staining Solution (BioLegend) and incubated for 15 min at room temperature in the dark. After adding 400 µL of Annexin V Binding Buffer (BioLegend), we analyzed cell apoptosis using flow cytometry.

At 72 h post-transfection, 1 × 10⁶ transfected cells were washed 2 times with cold BioLegend cell staining buffer (420201, BioLegend, San Diego, CA, USA) and resuspended in 100 µL of Annexin V Binding buffer (42220, BioLegend). The whole suspension was then treated with 5 µL of APC Annexin V (640920, BioLegend) and 10 µL of Propidium Iodide solution (421301, BioLegend) and incubated in the dark for 15 min. After that, 400 µL of Annexin V Binding buffer (42220, BioLegend) was added, and the apoptotic cells were measured by BD FACSCalibur™ Flow Cytometer (BD Biosciences, San Jose, CA, USA).
For cell cycle progression, 1 × 10⁶ transfected cells were washed 1 time with cold PBS. Then, 1 mL of cold 70% ethanol was added dropwise to the cell pellet and fixed at 4 °C for 30 min. After that, the fixed cells were collected and resuspended in 1 mL of PBS. The fixed cells were then treated with 50 µL of 100 µg/mL RNase A (Thermo Scientific, Boston, MA, USA), 425 µL of cell staining buffer (420201, BioLegend), and 25 µL of Propidium Iodide solution (421301, BioLegend). Finally, cell cycle profile analysis was measured by using BD FACSCalibur™ Flow Cytometer (BD Biosciences).

Apoptosis was detected using an annexin V Alexa Fluor 647 conjugate antibody (640912, BioLegend, Cambridge, UK) following the manufacturer's protocol. Briefly, 2 × 10⁶ cells were harvested and resuspended in 500 μl of annexin V binding buffer (BioLegend) either with or without annexin V antibody (antibody was diluted 1:100 in annexin V binding buffer). After 1 h of incubation on ice, 10 μl of a 200 μg/ml DAPI solution was added to cells before the analysis using a BD LSRII flow cytometer.

MCF-7 and MDA-MB231 cells were cultured as indicated above with G4 ligands for three and six days. After the incubation with G4 ligands and IPA as control compound, cells were detached with trypsin or PBS/EDTA for Annexin V staining and washed with PBS. For cell cycle, cells were fixed in 70% (v/v) ethanol at least overnight. Pellets were washed with PBS and re-suspended in PBS containing RNaseA (Roche) (0.4 U) and PI (0.015 mol/L), incubated for 20 min at room temperature and analyzed for emission in the FL3 channel. To remove artifacts, doublets and aggregates, an electronic doublet discrimination was performed using the area versus width of the fluorescence (FL3) pulse. For apoptosis induction, collected cells were washed with Annexin V binding buffer (Biolegend, San Diego, CA, USA). The pellet was re-suspended in 50 μL of Annexin V binding buffer containing fluorescein isothiocyanate (FITC)-conjugated Annexin V (1:40 Biolegend, #640906). After 15 min of incubation at room temperature, 250 μL PI solution (0.0015 mol/L in PBS) was added to each sample just before analysis. For calreticulin staining, cells were collected washed with PBS and stained with anti-calreticulin-PE conjugated (1:100, Enzo Life Sciences, New York, NY, USA) for 15 min on ice. After the incubation, cells were washed with PBS and analyzed by flow cytometry.

Apoptotic cells with externalised phosphatidylserine (PS) were detected using annexin V, a protein that binds to PS in a calcium-dependent way [57 (link)]. The cells were washed once with PBS, followed by a wash with annexin V binding buffer (BioLegend). The cells were then stained with 0.5 μg/ml annexin V-PE (BioLegend) and 1 μg/ml LIVE/DEAD fixable near-IR dye in annexin V binding buffer for 15 minutes. They were subsequently washed once with annexin V binding buffer containing 5% FCS and fixed with 4% formaldehyde diluted 1:1 with annexin V binding buffer. Finally, the cells were washed twice and immediately acquired on a BD LSRFortessa after resuspending in annexin V binding buffer containing 5% FCS. All washes and incubations were performed at room temperature. Data were analysed using FlowJo.