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CytoTell™ Violet 500

Cell proliferation assay with CytoTell™ Violet 500.  Jurkat cells (~2×10<sup>6</sup> cells/mL) were stained with CytoTell™ Violet 500 on Day 0. The cells were passed serially at 1:1 ratio on the day specified. Fluorescence intensity was measured with ACEA NovoCyte 3000 flow cytometer with Pacific Orange channel. Successive generations were represented by different colors.
Cell proliferation assay with CytoTell™ Violet 500.  Jurkat cells (~2×10<sup>6</sup> cells/mL) were stained with CytoTell™ Violet 500 on Day 0. The cells were passed serially at 1:1 ratio on the day specified. Fluorescence intensity was measured with ACEA NovoCyte 3000 flow cytometer with Pacific Orange channel. Successive generations were represented by different colors.
Ordering information
Price ()
Catalog Number22248
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 weight557.02
Spectral properties
Excitation (nm)415
Emission (nm)499
Storage, safety and handling
Intended useResearch Use Only (RUO)

OverviewpdfSDSpdfProtocol


Molecular weight
557.02
Excitation (nm)
415
Emission (nm)
499
Flow cytometry combined with fluorescence staining is a powerful tool to analyze heterogeneous cell populations. Among all the existing fluorescent dyes CFSE is the preferred cell proliferation indicator that is widely used for live cell analysis. However, it is impossible to use CFSE and its fluorescein analogs for GFP-transfected cells or for the applications where a FITC-labeled antibody is used since CFSE and its fluorescein analogs have the excitation and emission spectra almost identical to GFP or FITC. CytoTell™ dyes are well excited at major laser lines such as 405 nm, 488 nm or 633 nm with multicolor emissions. CytoTell™ dyes have minimal cytotoxicity, and are used for the multicolor applications with either GFP cell lines or FITC-labeled antibodies since they have either excitation or emission spectra distinct from fluorescein. CytoTell™ Violet 500 is a violet lase- excitable green fluorescent dye that stains cells evenly. As cells divide, the dye is distributed equally between daughter cells that can be measured as successive halving of the fluorescence intensity of the dye. Cells labeled with CytoTell™ Violet 500 may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ Violet 500 has a peak excitation of 405 nm and can be excited by the violet (405 nm) laser line. It has a peak emission of 500 nm and can be detected with a 500/20 band pass filter (equivalent to BD Horizon® V500), making it compatible with applications that utilize GFP or FITC antibodies for multicolor cell analysis.

Platform


Flow cytometer

Excitation405 nm laser
Emission525/50 nm filter
Instrument specification(s)Pacific Orange channel

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds
  2. Add 1X dye working solution
  3. Incubate dyes with cells at room temperature or 37 oC for 10 to 30 minutes
  4. Remove the dye working solution
  5. Analyse with flow cytometer with appropriate filter set

Important notes
Bring all the kit components at room temperature before starting the experiment. Note: The CytoTell™ dyes are lyophilized powders. They should be stable for at least 6 months if store at -20 °C, protecting from light, and avoiding freeze/thaw cycles.

Product Number

Indicator

Size

Ex/Em (nm)

Excitation Source

22240

CytoTell™ UltraGreen

500 tests

492/519

488 nm (Blue Laser)

22241

CytoTell™ UltraGreen

1000 tests

492/519

488 nm (Blue Laser)

22248

CytoTell™ Violet 500

500 tests

415/499

405 nm (Violet Laser)

22251

CytoTell™ Blue

500 tests

403/454

405 nm (Violet Laser)

22252

CytoTell™ Blue

1000 tests

403/454

405 nm (Violet Laser)

22253

CytoTell™ Green

500 tests

511/525

488 nm (Blue Laser)

22254

CytoTell™ Green

1000 tests

511/525

488 nm (Blue Laser)

22255

CytoTell™ Red 650

500 tests

628/643

633 nm (Red Laser)

22256

CytoTell™ Red 650

1000 tests

628/643

633 nm (Red Laser)

22257

CytoTell™ Orange

500 tests

542 /556

488 nm (Blue Laser)
531 nm (Green Laser)

22258

CytoTell™ Orange

1000 tests

542 /556

488 nm (Blue Laser)
531 nm (Green Laser)

22261

CytoTell™ Red 590

500 tests

560 /574

488 nm (Blue Laser)
531 nm (Green Laser)

22262

CytoTell™ Red 590

1000 tests

560 /574

488 nm (Blue Laser)
531 nm (Green Laser)

PREPARATION OF STOCK SOLUTION

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.

CytoTell™ dye stock solution (500X):
Add 500 µL DMSO into the dye powder vial, mix it well by vortexing to have a stock solution (500X). Note: The stock solution should be used promptly; any remaining solution should be aliquoted and frozen at < - 20 oC. Avoid repeated freeze-thaw cycles, and protect from light.

PREPARATION OF WORKING SOLUTION

CytoTellTM dye working solution (1X):
Dilute the 500X DMSO stock solution at 1 to 500 in Hanks and 20 mM Hepes buffer (HHBS) or the buffer of your choice, pH 7 (such as 1 µL of 500X DMSO stock solution to 500 µL buffer) right before use. Mix them well by vortexing. Note: The final concentration of the dye working solution should be empirically determined for different cell types and/or experimental conditions. It is recommended to test at the concentrations that are at least over ten fold range. Such as CytoTell™ Red might use much less amount in some cell types than the recommend concentrations.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat cells with test compounds for a desired period of time.

  2. Centrifuge the cells to get 1-5 × 105 cells per tube.

  3. Resuspend cells in 500 µL of the CytoTell™ dye working solution. Optional: One can add the 500X DMSO stock solution into the cells directly without medium removing (such as, add 1 µL500X DMSO stock solution into 500 µL cells)

  4. Incubate cells with a dye solution at room temperature or 37 °C for 10 to 30 minutes, protected from light.

  5. Remove the dye working solution from the cells, wash the cells with HHBS or buffer of your choice. Resuspend cells in 500 µL of pre-warmed HHBS or medium to get 1-5 × 105 cells per tube.

  6. Monitor the fluorescence change at respected Ex/Em (see Table 1) with a flow cytometer or a fluorescence microscope.

Calculators


Common stock solution preparation

Table 1. Volume of appropriate solvent needed to reconstitute specific mass of CytoTell™ Violet 500 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 mM179.527 µL897.634 µL1.795 mL8.976 mL17.953 mL
5 mM35.905 µL179.527 µL359.054 µL1.795 mL3.591 mL
10 mM17.953 µL89.763 µL179.527 µL897.634 µL1.795 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)415
Emission (nm)499

Citations


View all 29 citations: Citation Explorer
Cardiomyocyte cell cycle dynamics and proliferation revealed through cardiac-specific transgenesis of fluorescent ubiquitinated cell cycle indicator (FUCCI)
Authors: Alvarez, R., Jr., Wang, B. J., Quijada, P. J., Avitabile, D., Ho, T., Shaitrit, M., Chavarria, M., Firouzi, F., Ebeid, D., Monsanto, M. M., Navarrete, N., Moshref, M., Siddiqi, S., Broughton, K. M., Bailey, B. A., Gude, N. A., Sussman, M. A.
Journal: J Mol Cell Cardiol (2019): 154-164
In Vivo Fluorescence Microendoscopic Monitoring of Stent-Induced Fibroblast Cell Proliferation in an Esophageal Mouse Model
Authors: Jun, E. J., Song, H. Y., Park, J. H., Bae, Y. S., Paulson, B., Lee, S., Cho, Y. C., Tsauo, J., Kim, M. T., Kim, K. Y., Yang, S. G., Kim, J. K.
Journal: J Vasc Interv Radiol (2018): 1756-1763
Visualizing Breast Cancer Cell Proliferation and Invasion for Assessing Drug Efficacy with a Fluorescent Nanoprobe
Authors: Luan, M., Yu, L., Li, Y., Pan, W., Gao, X., Wan, X., Li, N., Tang, B.
Journal: Anal Chem (2017): 10601-10607
Monitoring cell proliferation in vitro with different cellular fluorescent dyes
Authors: Zolnierowicz, J., Ambrozek-Latecka, M., Kawiak, J., Wasilewska, D., Hoser, G.
Journal: Folia Histochem Cytobiol (2013): 193-200
A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution
Authors: Begum, J., Day, W., Henderson, C., Purewal, S., Cerveira, J., Summers, H., Rees, P., Davies, D., Filby, A.
Journal: Cytometry A (2013): 1085-95
Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice
Authors: Vacaru, A. M., Isern, J., Fraser, S. T., Baron, M. H.
Journal: Genesis (2013): 751-62
Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation
Authors: He, H., Zhan, Y., Zhang, Y., Zhang, J., He, L.
Journal: Luminescence (2012): 310-4
Long-term monitoring of live cell proliferation in presence of PVP-Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE
Authors: Penjweini, R., Loew, H. G., Hamblin, M. R., Kratky, K. W.
Journal: J Microsc (2012): 100-8
Assessment of cell proliferation with resazurin-based fluorescent dye
Authors: Czekanska, E. M.
Journal: Methods Mol Biol (2011): 27-32
The proliferation and differentiation of osteoblasts in co-culture with human umbilical vein endothelial cells: An improved analysis using fluorescence-activated cell sorting
Authors: Zhang, Y., Schedle, A., Matejka, M., Rausch-Fan, X., Andrukhov, O.
Journal: Cell Mol Biol Lett (2010): 517-29

Application notes


Annexin V