CytoTell™ Violet 500

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Telephone	1-800-990-8053
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Email	sales@aatbio.com
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Physical properties

Molecular weight	557.01
Solvent	DMSO

Spectral properties

Excitation (nm)	415
Emission (nm)	499

Storage, safety and handling

Intended use	Research Use Only (RUO)
Storage	Freeze (< -15 °C); Minimize light exposure

Overview SDS Protocol

Molecular weight

557.01

Excitation (nm)

415

Emission (nm)

499

Flow cytometry combined with fluorescence staining is a powerful tool for analyzing heterogeneous cell populations. CFSE is the preferred cell proliferation indicator widely used for live cell analysis among all the existing fluorescent dyes. However, it is impossible to use CFSE and its fluorescein analogs for GFP-transfected cells or for 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 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 laser-excitable green fluorescent dye that stains cells evenly. As cells divide, the dye is distributed equally between daughter cells, which can be measured as a successive halving of the fluorescence intensity of the dye. Cells labeled with CytoTell™ Violet 500 may be fixed and permeabilized to analyze 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 bandpass filter (equivalent to BD Horizon® V500), making it compatible with applications that utilize GFP or FITC antibodies for multicolor cell analysis.

Platform

Flow cytometer

Excitation	405 nm laser
Emission	525/50 nm filter
Instrument specification(s)	Pacific Orange channel

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds
Add 1X dye working solution
Incubate dyes with cells at room temperature or 37 ^oC for 10 to 30 minutes
Remove the dye working solution
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

CytoTell^TM 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

Treat cells with test compounds for a desired period of time.
Centrifuge the cells to get 1-5 × 10⁵ cells per tube.
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)
Incubate cells with a dye solution at room temperature or 37 °C for 10 to 30 minutes, protected from light.
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.
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 DMSO 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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	179.53 µL	897.65 µL	1.795 mL	8.976 mL	17.953 mL
5 mM	35.906 µL	179.53 µL	359.06 µL	1.795 mL	3.591 mL
10 mM	17.953 µL	89.765 µL	179.53 µL	897.65 µL	1.795 mL

Molarity calculator

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Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Excitation (nm)	415
Emission (nm)	499

Product Family

Name	Excitation (nm)	Emission (nm)
CytoCalcein™ Violet 500 Excited at 405 nm	420	505
ThiolTrace™ Violet 500	415	499

Images

Figure 1. Cell proliferation assay with CytoTell™ Violet 500. Jurkat cells (~2×10⁶ 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.

Citations

View all 30 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