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Molecular Probes™ LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation
Description
The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit is used to determine the viability of cells prior to the fixation and permeabilization required for intracellular antibody staining or prior to elimination of biohazardous materials using formaldehyde fixation. This kit has been optimized and validated for use with a violet laser flow cytometer.
- Stable—dyes are freeze dried in separate vials to maintain stability
- Robust—staining pattern is the same before and after fixation
- Low compensation—minimal spectral overlap between other fluorophores
Stable
The product is sold in tubes with DMSO and in lyophilized flow tubes and plates which require no DMSO. Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use material once rehydrated.
Robust
Dead cell discriminator stains can lose sensitivity after treatment with fixatives such as formaldehyde or ethanol-based fixation methods required for intracellular phosphorylation studies. The LIVE/DEAD™ Fixable Aqua Stain is an amine reactive dye that binds covalently to intracellular and extracellular amines, and the staining pattern is preserved following formaldehyde fixation.
Low compensation
The LIVE/DEAD™ Fixable Aqua Stain was selected based on its fluorescent properties to minimize compensation between other violet dyes and dyes that excite off of the 488 nm blue laser. The aqua-fluorescent reactive dye has an excitation maximum of ∼375 nm, but it is excited well with the 405 nm violet laser, and it has an emission maxima of ∼512 nm, so it can be collected in the second channel on most violet laser flow cytometers.
How it works
In cells with compromised membranes, the dye reacts with free amines both in the cell interior and on the cell surface, yielding intense fluorescent staining. In viable cells, the dye's reactivity is restricted to the cell-surface amines, resulting in less intense fluorescence. The difference in intensity is typically greater than 50-fold between live and dead cells, allowing for easy discrimination.
Colors available
LIVE/DEAD™ Fixable Dead Cell Stains are available in a wide variety of colors to meet your multi-color panel needs.
Specifications
Specifications
| Cell Permeability | Impermeant |
| Cell Type | Eukaryotic Cells |
| Color | Aqua |
| Content And Storage | Contains 10 vials of LIVE/DEAD™ fixable dead cell stain and 500 μL DMSO. Store at -20°C. |
| Description | LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation |
| Detection Method | Fluorescence |
| For Use With (Application) | Viability Assay |
| For Use With (Equipment) | Flow Cytometer |
| Product Type | Stain |
| Dye Type | LIVE/DEAD™ Fixable Aqua Dead Cell Stain |
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Frequently Asked Questions (FAQs)
There are two easy options. One is to heat-inactivate the cells by placing at 60 degrees C for 20 minutes. The second is to subject the cells to 70% ethanol. Alcohol-fixed cells can be stored indefinitely in the freezer until use, potentially up to several years.
Centrifuge cells, pellet, and remove supernatant.
Fix cells: Add 10 mL ice cold 70% ETOH to a 15 mL tube containing the cell pellet, adding dropwise at first while vortexing, mix well.
Store in freezer until use.
When ready to use, wash twice and resuspend in buffer of choice.
The LIVE/DEAD Fixable kits for flow cytometry analysis are compatible with fixation. These kits use amine-reactive cell-impermeant dyes that stain the cell surface of live cells and also the cytosol of dead cells-live cells are dim and dead cells are bright. Since the dye is covalently bound to the cells, it will be retained after fixation. Unfortunately, this method does not work well for imaging-based assays, as all cells are stained and it is difficult to distinguish bright dead cells from dim live cells with a microscope. Ethidium monoazide (EMA; Cat No. E1374) is a cell impermeant nucleic acid stain that can be applied to live cultures and stains only dead cells. After incubation and washing away unbound dye, the cells can be exposed to light to photoactivate EMA to crosslink to dead cell DNA. After crosslinking to dead cell DNA, the samples may be fixed and permeabilized.
Image-IT DEAD Green Viability Stain (Cat. No. I10291) for imaging and high-content screening (HCS) analysis is a live-cell impermeant DNA binding dye that is compatible with fixation and permeabilization with good retention up to 48 hours.
We also have a LIVE/DEAD Reduced Biohazard Cell Viability Kit (Cat. No. L7013) for imaging and flow analysis that contains two DNA binding dyes, SYTO 10 and Dead Red, that are sufficiently retained to be analyzed soon after 4% glutaraldehyde fixation.
Note: In general, DNA-binding dyes and calcein AM are not compatible with fixation, as these dyes are not covalently bound to components of the cell and will thus slowly diffuse out of cells after fixation, gradually staining all cells as dead.
For Research Use Only. Not for use in diagnostic procedures.