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Cell Meter™ BX520 fixable viability dye
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™ BX520 fixable cell stain is optimized to be excited with the blue laser at 488 nm with emission at 520 nm (FITC channel). Compared to other commercially similar viability dyes, this fixable viability dye is much more robust and stable.
Example protocol
AT A GLANCE
Protocol summary
- Prepare samples in HHBS (0.5 mL/assay)
- Add Cell Meter™ BX520 to the cell suspension
- Stain the cells at room temperature for 20 - 60 minutes
- Wash the cells
- Fix the cells (optional)
- Examine the sample with flow cytometer using 530/30 nm filter (FITC channel) and/or fluorescence microscope using FITC 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.
Note The unused Cell Meter™ BX520 stock solution should be divided into single use aliquots and stored at -20 °C. Avoid repeated freeze/thaw cycles.
Cell Meter™ BX520 stock solution (500X)
Add 200 µL of DMSO into the Cell Meter™ BX520 vial to make 500X stock solution.Note The unused Cell Meter™ BX520 stock solution should be divided into single use aliquots and stored at -20 °C. Avoid repeated freeze/thaw cycles.
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells as desired.
- Wash cells once with HHBS or the azide- and serum/protein-free buffer of your choice.
- Resuspend cells at 5 - 10 × 10 6/mL in HHBS or in the azide- and serum/protein-free buffer of your choice.
- Add 1 µL of 500X Cell Meter™ BX520 stock solution to 0.5 mL of cells/assay and mix it well.
- 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. - Wash cells twice and resuspend cells with HHBS or the buffer of your choice.
- Fix cells as desired (optional).
- Analyze cells with a flow cytometer using 530/30 nm filter (FITC channel) and/or fluorescence microscope using FITC filter set.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Cell Meter™ BX520 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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 113.41 µL | 567.048 µL | 1.134 mL | 5.67 mL | 11.341 mL |
| 5 mM | 22.682 µL | 113.41 µL | 226.819 µL | 1.134 mL | 2.268 mL |
| 10 mM | 11.341 µL | 56.705 µL | 113.41 µL | 567.048 µL | 1.134 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| Cell Meter™ BX590 fixable viability dye | 492 | 579 |
| Cell Meter™ BX650 fixable viability dye | 518 | 654 |
| Cell Meter™ IX830 fixable viability dye | 811 | 822 |
| Cell Meter™ RX660 fixable viability dye | 649 | 664 |
| Cell Meter™ RX700 fixable viability dye | 690 | 713 |
| Cell Meter™ RX780 fixable viability dye | 629 | 767 |
| Cell Meter™ VX450 fixable viability dye | 406 | 445 |
| Cell Meter™ VX500 fixable viability dye | 433 | 498 |
References
View all 50 references: Citation Explorer
CFSE dilution to study human T and NK cell proliferation in vitro.
Authors: Terrén, Iñigo and Orrantia, Ane and Vitallé, Joana and Zenarruzabeitia, Olatz and Borrego, Francisco
Journal: Methods in enzymology (2020): 239-255
Authors: Terrén, Iñigo and Orrantia, Ane and Vitallé, Joana and Zenarruzabeitia, Olatz and Borrego, Francisco
Journal: Methods in enzymology (2020): 239-255
Using Carboxy Fluorescein Succinimidyl Ester (CFSE) to Identify Quiescent Glioblastoma Stem-Like Cells.
Authors: Azari, Hassan and Deleyrolle, Loic P and Reynolds, Brent A
Journal: Methods in molecular biology (Clifton, N.J.) (2018): 59-67
Authors: Azari, Hassan and Deleyrolle, Loic P and Reynolds, Brent A
Journal: Methods in molecular biology (Clifton, N.J.) (2018): 59-67
Estimates and impact of lymphocyte division parameters from CFSE data using mathematical modelling.
Authors: Mazzocco, Pauline and Bernard, Samuel and Pujo-Menjouet, Laurent
Journal: PloS one (2017): e0179768
Authors: Mazzocco, Pauline and Bernard, Samuel and Pujo-Menjouet, Laurent
Journal: PloS one (2017): e0179768
Assessment of lymphocyte proliferation for diagnostic purpose: Comparison of CFSE staining, Ki-67 expression and 3H-thymidine incorporation.
Authors: Lašťovička, Jan and Rataj, Michal and Bartůňková, Jiřina
Journal: Human immunology (2016): 1215-1222
Authors: Lašťovička, Jan and Rataj, Michal and Bartůňková, Jiřina
Journal: Human immunology (2016): 1215-1222
Analysis of CFSE time-series data using division-, age- and label-structured population models.
Authors: Hross, Sabrina and Hasenauer, Jan
Journal: Bioinformatics (Oxford, England) (2016): 2321-9
Authors: Hross, Sabrina and Hasenauer, Jan
Journal: Bioinformatics (Oxford, England) (2016): 2321-9
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