CytoTell™ Orange

Name: CytoTell™ Orange
Brand: AAT Bioquest
SKU: 22257
Availability: InStock

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™ Red is a red 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. Up to 8 generations may be visualized. CytoTell™ Orange can also be used for long term tracking of labeled cells. Analysis using two-parameter plots may provide better resolution of each generation, especially between undivided cells and the first generation. Cells labeled with CytoTell™ Orange may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ Orange has a peak excitation of 542 nm and can be excited by the blue (488 nm) and green (532 nm) laser lines. It has a peak emission of ~560 nm and can be detected with a 575/26 band pass filter (equivalent to RPE).

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™ Orange 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	161.413 µL	807.063 µL	1.614 mL	8.071 mL	16.141 mL
5 mM	32.283 µL	161.413 µL	322.825 µL	1.614 mL	3.228 mL
10 mM	16.141 µL	80.706 µL	161.413 µL	807.063 µL	1.614 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

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)
CytoTell™ Blue	410	445
CytoTell™ Green	510	525

Citations

View all 8 citations: Citation Explorer

Inserting EF1$\alpha$-driven CD7-specific CAR at CD7 locus reduces fratricide and enhances tumor rejection
Authors: Jiang, Jie and Chen, Jiangqing and Liao, Chan and Duan, Yanting and Wang, Yajie and Shang, Kai and Huang, Yanjie and Tang, Yongming and Gao, Xiaofei and Gu, Ying and others,
Journal: Leukemia (2023): 1--11

In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag
Authors: Govindan, Subashika and Oberst, Polina and Jabaudon, Denis
Journal: Nature protocols (2018): 2297--2311

CXCL12--CXCR4 Axis Is Required for Contact-Mediated Human B Lymphoid and Plasmacytoid Dendritic Cell Differentiation but Not T Lymphoid Generation
Authors: Minami, Hirohito and Nagaharu, Keiki and Nakamori, Yoshiki and Ohishi, Kohshi and Shimojo, Naoshi and Kageyama, Yuki and Matsumoto, Takeshi and Sugimoto, Yuka and Tawara, Isao and Masuya, Masahiro and others, undefined
Journal: The Journal of Immunology (2017): ji1700054

Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells
Authors: Klöss, Volker and Grünvogel, Oliver and Wabnitz, Guido and Eigenbrod, Tatjana and Ehrhardt, Stefanie and Lasitschka, Felix and Lohmann, Volker and Dalpke, Alex and er H, undefined
Journal: Frontiers in Immunology (2017): 1238

Cooperation of innate immune cells during Hepatitis C virus infection
Authors: Klöss, Volker
Journal: (2017)

Page updated on December 17, 2024

Ordering information

Price
Unit size	500 Tests 2x500 Tests
Catalog Number	2225722258
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Physical properties

Molecular weight	619.53
Solvent	DMSO

Spectral properties

Excitation (nm)	541
Emission (nm)	560

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200