Green Live/Dead Stain

$95.00

Green Live/Dead Stain is a live-cell impermeant, green fluorescence-emitting DNA dye for viability, apoptosis and necrosis studies, and fixed cell nuclear counterstaining. This product binds to dsDNA/nuclei of necrotic or permeabilized cells and can be used in combination with live cell dyes for live/dead discrimination. Analyze samples using a flow cytometer or fluorescence microscope.

Catalog # Product Size Quantity Price
6342 50 µL $95.00
Catalog #: 6342 Categories: ,

Green Live/Dead Stain is a membrane impermeant fluorescent vital stain for differentiating live and dead cells.

This green Live/Dead Stain is a vital dye that exhibits intact cell membrane exclusion properties analogous to the popular red fluorescing vital dyes, Propidium Iodide (PI), 7-aminoactinomycinD (7-AAD), and DRAQ7™. Like the red fluorescing DNA dyes, Green Live/Dead Stain is excluded from intact, healthy cells due to its polar nature. In the presence of cells exhibiting compromised membrane integrity, Green Live/Dead Stain penetrates the cell and nuclear membrane barriers and intercalates tightly to DNA in a manner analogous to PI and 7-AAD. When bound to DNA, it acquires a greatly enhanced fluorescence potential (>2000X) in the green emission range. These important vital dye properties enable Green Live/Dead Stain to be used in flow cytometry-based protocols and microscopy analysis techniques to assess the percentage of late apoptotic, necrotic, and membrane-compromised cells within a sample cell population. The green fluorescence emission permits new combinations of dyes to be used in the understanding of cell death and apoptosis, such as use with red SR-FLICA® assays, far-red FLICA 660 caspase assays, and other visible range fluor-tagged ligands.

Green Live/Dead Stain only stains nuclei in dead and permeabilized cells. Green Live/Dead Stain does not enter intact, live cells. Cells may be fixed or permeabilized for use of Green Live/Dead Stain as a nuclear counterstain. With negligible toxicity, Green Live/Dead Stain can be used in long-term viability assays to assess cytotoxicity and apoptosis.

For microscopy applications, Green Live/Dead Stain will function as a DNA counterstain for fixed cell and tissue samples in IF, IHC, high content screening, and cell-based assays.

Flow Cytometry
1. Each 50 µL vial is sufficient for 1000 flow cytometry tests.
2. Spin thawed vial briefly in a microcentrifuge to remove any reagent that may have become trapped in the cap.
3. Dilute Green Live/Dead Stain concentrated stock solution (500 µM) 1:100 in PBS to prepare a 5000 nM working solution. For example, add 10 µL stock concentrate to 990 µL PBS. Prepare only what is needed for the experiment. Working solution should be used immediately, and any remaining diluted solution should be discarded.
4. Spike samples with a 1:100 dilution of the 5000 nM working solution. For example, spike 0.495 mL samples with 5 µL working solution.
5. Incubate samples for ~10 minutes at room temperature, protected from light.
6. Analyze with a flow cytometer using a blue laser at 488 nm and a 530/30 (FL1) emission filter setting, or similar.

Fluorescence Microscopy
1. Each 50 µL vial is sufficient for 100 microtiter plate wells.
2. Spin thawed vial briefly in a microcentrifuge to remove any reagent that may have become trapped in the cap.
3. Dilute Green Live/Dead Stain concentrated stock solution (500 µM) 1:100 in PBS to prepare a 5000 nM working solution. For example, add 10 µL stock concentrate to 990 µL PBS. Prepare only what is needed for the experiment. Working solution should be used immediately, and any remaining diluted solution should be discarded.
4. Spike samples with a 1:10 dilution of the 5000 nM working solution. For example, spike .45 mL samples with 50 µL working solution.
5. Incubate samples for ~10 minutes at room temperature, protected from light.
6. When bound to DNA, the peak absorption of Green Live/Dead Stain is 495 nm and the maximum emission is 512 nm. Visualize with a fluorescence microscope using optical filters that best approximate these settings.

Green Live/Dead Stain and FLICA® 660 Poly Caspase Assay were used to assess cell death in staurosporine-treated Jurkat cells. Following a four hour induction period, treated cells were stained with the reagents and imaged witha Nikon E800 microscope (DIC overlay image, left). The majority of cells were in the early stages of apoptosis (active caspases stained red with FLICA® 660). One cell in this field of view (bottom right corner) exhibited both red and green fluorescence, indicating the loss of membrane integrity that is characteristic of late-stage apoptosis.

CellMetrix_Green_Live-Dead_Stain_FLICA_660_Pos_Fig_2CellMetrix_Green_Live-Dead_Stain_FLICA_660_Neg_Fig_2

Jurkat cells were exposed to 3% formaldehyde for 30 minutes to create a cell population with a mix of live and dead cells. Following the formaldehyde treatment, cells were stained with Green Live/Dead Stain and then imaged with a Nikon E800 microscope (DIC overlay image is shown below). Cells with compromised membranes (for example, necrotic, dying, or dead cells) stained green, while cells with intact membranes exclude the dye and remain unstained.

Necrotic_or_Dead_Cell_Staining_CellMetrix_Green_Live-Dead_Stain

• Target: dead cells, necrosis
• Excitation/Emission: 495 nm / >512 nm, when bound to nucleic acids
• Supplied at: 500 µM
• Method of Analysis: Flow Cytometer, Fluorescence Microscope
• Types of Samples: cell culture
• Storage: -20°C
• Shipping: Ships overnight (domestic)

Documentation

Manual
Safety Data Sheet