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Cell Meter™ BX650 fixable viability dye

Detection of Jurkat cell viability by Cell Meter™ fixable viability dye. Jurkat cells were treated and stained with Cell Meter™ BX650 (Cat#22520), and then fixed in 3.7% formaldehyde and analyzed by flow cytometry.  The dead cell population (Blue peak)  is easily distinguished from the live cell population (Red peak)  with PerCP channel, and nearly identical results were obtained before and after fixation.
Detection of Jurkat cell viability by Cell Meter™ fixable viability dye. Jurkat cells were treated and stained with Cell Meter™ BX650 (Cat#22520), and then fixed in 3.7% formaldehyde and analyzed by flow cytometry.  The dead cell population (Blue peak)  is easily distinguished from the live cell population (Red peak)  with PerCP channel, and nearly identical results were obtained before and after fixation.
Ordering information
Price ()
Catalog Number22520
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight844.83
SolventDMSO
Spectral properties
Excitation (nm)518
Emission (nm)654
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
844.83
Excitation (nm)
518
Emission (nm)
654
Discrimination and exclusion of dead cells from live cells allows cleaner separation and identification of cell populations. Cell Meter™ fixable viability dyes are a large family of cell-impermeable fluorescent viability dyes that are optimized to match the major excitation lasers of common flow cytometers, such as 350, 405, 488, 633 and 647 nm. These dyes are impermeant to live cells but permeant to cells with compromised membranes. They irreversibly react with amine- and thiol-containing proteins and other cellular components. Since dead or fixed cells with a compromised membrane more readily react with Cell Meter™ fixable cell stains, thus stain brighter than live cells with an intact membrane, these dyes can be used to assess live vs. dead status of mammalian cells. There are a few factors to be considered when using these dyes, e.g., the titration of each dye to ensure that live cells have minimal to no staining. Cell Meter™ BX650 fixable cell stain is optimized to be excited with the blue laser at 488 nm with emission at 650 nm. Compared to other commercially similar viability dyes, this fixable viability dye is much more robust and stable.

Platform


Flow cytometer

Excitation488 nm laser
Emission695/40 nm filter
Instrument specification(s)PerCP channel

Fluorescence microscope

ExcitationTexasRed filter set
EmissionTexasRed filter set
Recommended plateBlack wall/clear bottom

Example protocol


AT A GLANCE

Protocol summary
  1. Prepare samples in HHBS (0.5 mL/assay)
  2. Add Cell Meter™ BX650 to the cell suspension
  3. Stain the cells at room temperature for 20 - 60 minutes
  4. Wash the cells
  5. Fix the cells (optional)
  6. Examine the sample with flow cytometer using 695/40 nm filter (PerCP channel) and/or fluorescence microscope using Texas Red filter set 

Important
Thaw at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Cell Meter™ BX650 stock solution (500X)
Add 200 µL of DMSO into the Cell Meter™ BX650 vial to make 500X stock solution.
Note     The unused Cell Meter™ BX650 stock solution should be divided into single use aliquots and stored at -20 °C. Avoid repeated freeze/thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat cells as desired.
  2. Wash cells once with HHBS or the azide- and serum/protein-free buffer of your choice.
  3. Resuspend cells at 5 - 10 × 106/mL in HHBS or in the azide- and serum/protein-free buffer of your choice.
  4. Add 1 µL of 500X Cell Meter™ BX650 stock solution to 0.5 mL of cells/assay and mix it well.
  5. Incubate at room temperature, 5 % CO2 incubator for 20 - 60 minutes, protected from light.
    Note     The optimal stain concentrations and incubation time should be experimentally determined for different cell lines.
  6. Wash cells twice and resuspend cells with HHBS or the buffer of your choice.
  7. Fix cells as desired (optional).
  8. Analyze cells with a flow cytometer using 695/40 nm filter (PerCP channel) and/or fluorescence microscope using Texas Red filter set. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cell Meter™ BX650 fixable viability dye to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM118.367 µL591.835 µL1.184 mL5.918 mL11.837 mL
5 mM23.673 µL118.367 µL236.734 µL1.184 mL2.367 mL
10 mM11.837 µL59.184 µL118.367 µL591.835 µL1.184 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)518
Emission (nm)654

References


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Fluorescence-based method is more accurate than counting-based methods for plotting growth curves of adherent cells.
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Journal: Annals of clinical and laboratory science (2019): 740-747
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Journal: PloS one (2019): e0221878
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Journal: Cytometry. Part B, Clinical cytometry (2018): 363-368
Delineating the distinct role of AKT in mediating cell survival and proliferation induced by CD154 and IL-4/IL-21 in chronic lymphocytic leukemia.
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BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity.
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Journal: Toxicology and applied pharmacology (2016): 56-67