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  6. Molecular Probes™ LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit, for 405 nm excitation

Molecular Probes™ LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit, for 405 nm excitation

Product Code. 10645203
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Product Code. 10645203 Supplier Molecular Probes™ Supplier No. L34955

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Description

The LIVE/DEAD™ Fixable Violet 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.

  • 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
  • Kit has been optimized and validated for use with a violet laser flow cytometer
  • Conveniently packaged in 40-test vials to help ensure the stability and performance of the dye over time
  • Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use the vial once rehydrated
  • If this is not possible, aliquot the vials in small volumes and store at -80°C, avoiding freeze-thaw cycles
  • LIVE/DEAD™ Fixable Dead Cell Stains are available in a wide variety of colors to meet multi-color panel needs

Low compensation

  • The LIVE/DEAD™ Fixable Violet 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 violet-fluorescent reactive dye has an excitation maximum of ∼416 nm and is excited well with the 405 nm violet laser
  • It has an emission maxima of ∼451 nm, so it can be collected in the first channel on most violet laser flow cytometers
TRUSTED_SUSTAINABILITY

Specifications

Cell Permeability Impermeant
Cell Type Eukaryotic Cells
Color Violet
Content And Storage Contains 5 vials of LIVE/DEAD™ fixable dead cell stain and 500 μL DMSO.

Store at -20°C.
Description LIVE/DEAD™ Fixable Violet 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 Violet Dead Cell Stain
Emission 451
Excitation Wavelength Range 416 nm
Form Solid
Format Tube(s)
Product Line LIVE/DEAD
Quantity 200 Assays
Shipping Condition Room Temperature
Solubility DMSO (Dimethylsulfoxide)
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Frequently Asked Questions (FAQs)

I need to use a dead cell control for my viability assay. Do you have a protocol for killing cells for this?

Heat killing is commonly used. Place your cells in a tube in buffer and heat at 60oC 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 -20oC until needed, at which point you wash out the ethanol and replace with buffer.
Regarding the LIVE/DEAD Fixable Dead Cell Stain Kits, which can discriminate between live and dead cells using flow cytometry with one emission wavelength. Can these kits be used with microscopy?

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.
How do I prepare dead cell controls for LIVE/DEAD cell viability assays?

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.
Which cell viability kits are compatible with fixation?

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.

Why do I need to include a viability stain in my assays?

Many antibodies and stains will label dead cells. This will give you misleading data if you do not exclude the dead cells from your analysis. Of course, if you are labeling fixed cells, they are already dead and you do not need a viability stain. However, if you label your cells prior to fixation, then you need to use one of the LIVE/DEAD Fixable Dead Cell Stains.

What is the smallest size that I can detect with the Attune NxT Acoustic Focusing Cytometer?

The smallest size that you can detect with the Attune NxT Acoustic Focusing Cytometer is 0.5 µm.
What is the Attune NxT Autosampler?

The Attune NxT Autosampler, an optional accessory for the Attune NxT Acoustic Focusing Cytometer, enables rapid processing of up to 384 samples. It has broad compatibility with different plate formats, both 96- and 384-well plates. It has an intelligent probe designed to minimize clogging and carryover (<0.5%) and to prevent damage to the instrument. It mixes by aspiration rather than shaking to ensure homogeneity of the sample and maintain cell viability. Is performs automated cleaning as part of the shutdown process of the Attune NxT Cytometer. It provides consistent data regardless of sampling method (tube vs. plate) and collection rate.
What are the advantages of acoustic-assisted hydrodynamic focusing in flow cytometry?

-Modular design - Multiple configurations available - field upgradable.
-Save time - 10X faster speeds with no loss in data quality.
-Simplified sample prep - No wash, no lyse options, non-clogging fluidics.
-Enables unique applications - Complex protocols on a broad range of cell types and samples.
How is the Attune NxT Acoustic Focusing Cytometer different from traditional flow cytometers?

With the option to be configured with up to 4 lasers and 14 colors for multi-parameter analysis the Attune NxT Acoustic Focusing Cytometer was designed as a modular system to fit most experimental needs and lab budgets. The novel design of the optical path helps ensure precise fixed alignment of four spatially separated lasers onto the sample stream enabling consistency in data over time, superior performance, and superior reliability. The instrument can be configured with up to 4 solid-state lasers (405 nm, 488 nm, 561 nm, and 637 nm) with flat top beam profiles.

The Attune NxT Flow Cytometer's acoustic focusing uses ultrasonic radiation pressure (> 2 MHz) to transport particles into the center of the sample stream. This pre-focused stream is then injected into the sheath stream, which supplies an additional hydrodynamic pressure to the sample. The combination of these two forces- termed acoustic-assisted hydrodynamic focusing-results in a narrow core stream and uniform laser illumination, regardless of the sample input rate. In traditional cytometers that rely solely on hydrodynamic focusing, the sample core widens to accommodate the increases in flow rate, which results in less uniform laser light illumination.
What is flow cytometry?

Cytometry is the measurement of physical or chemical characteristics of cells or particles. Flow cytometry measures these characteristics of cells or particles as they individually pass lasers in a flow cytometer instrument. Flow cytometry is performed on single cells, providing discrete measurements for each cell in the sample. It also provides a statistical distribution of the measured characteristics of the sample.

A flow cytometer is made up of three subsystems: fluidics, optics, and electronics. Fluidics moves the cells and introduces them for interrogation. Optics generates and collects the light signals. Electronics converts the optical signals to proportional electronic signals for computer analysis.

For Research Use Only. Not for use in diagnostic procedures.

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