LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation

Heat killing is commonly used. Place your cells in a tube in buffer and heat at 60^oC for 20 minutes. You can also kill your cells by fixing them with ice cold 70% ethanol for 15 minutes. The ethanol-killed cells can then be stored at -20^oC until needed, at which point you wash out the ethanol and replace with buffer.

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This dye gives a dim surface label for live cells, but is internalized and gives a brighter signal for dead cells. Flow cytometry is a very sensitive technique and can easily distinguish between the two populations. Microscopy is not as sensitive and may not be able to distinguish the cells because of a less sensitive detector.

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Once reconstituted, the Live/Dead fixable dyes are guaranteed to be stable at -20 degrees C for a few weeks. We have not tested storage conditions beyond this time frame. The dye breaks down in water so the condition of the DMSO that is used for resuspension is the most important determinant.

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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.

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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.

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