BD Via-Probe™ Green Nucleic Acid Stain

Panel 1 - Flow cytometric analysis of HeLa cell viability. Cells from the human HeLa (Cervical adenocarcinoma, ATCC CCL 2) cell line were heat-killed by incubation at 60°C (45 min), cooled to room temperature, and mixed with an equal number of untreated viable HeLa cells. Cells were then stained with BD Via-Probe™ Green Nucleic Acid Stain (Cat. No. 565799/565802 ; 10 nM) and then acquired by flow cytometry. Morphologically live (red solid line histogram) and dead (blue dashed line histogram) HeLa cells were gated based on their distinct forward and side light-scattering characteristics and can be clearly distinguished based on staining intensity of BD Via-Probe™ Green. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System. Panel 2 - Two-color flow cytometric analysis of mouse thymocyte viability. BALB/c thymocytes were left untreated (Left Plot) or stimulated with Purified NA/LE Hamster Anti-Mouse CD95 antibody (Cat. No. 554254; Right Plot) for 4 hours. Cells were harvested, stained with BD Horizon™ BV421 Annexin V (Cat. No. 563973) and BD Via-Probe™ Green Nucleic Acid Stain (5 nM), and analyzed by flow cytometry. Anti-CD95-stimulated cells show an increase in Annexin V-positive and BD Via-Probe™ Green-positive cells, indicating an increase in apoptosis and cell death. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System.

Panel 1 - Flow cytometric analysis of HeLa cell DNA content. HeLa cells in log phase growth were harvested, fixed, and permeabilized using 70% ice-cold ethanol while vortexing. Cells were resuspended in DPBS with 1 μ M BD Via-Probe™ Green Nucleic Acid Stain and 0.25 μ g/mL RNAse A (Sigma, Cat. No. R6513) and acquired by flow cytometry at a low flow rate using a BD LSRFortessa™ Cell Analyzer System. DNA histograms were deconvoluted by FlowJo™ software into G0/G1, S, and G2/M populations. Please note that for DNA content analysis, this dye is also compatible with BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725). Panel 2 - Immunofluorescent staining of HeLa cells. HeLa cells were seeded into a 96-well imaging plate at ~10,000 cells/well. After overnight incubation, the cells were fixed in BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and then stained with Alexa Fluor® 647 Mouse Anti-Actin antibody (Cat. No. 558624; pseudocolored red). Cell nuclei were counterstained with BD Via-Probe™ Green Nucleic Acid Stain (pseudocolored green) with 0.25 mg/mL RNAse A (Sigma Aldrich, Cat. No. R6513). The images were captured on a Molecular Devices ImageXpress® Micro XLS using a 20× objective and merged using Molecular Devices MetaXpress® software. Green Nucleic Acid Stain is also compatible with Saponin and Triton™ X-100 fix/perm protocols.