CytoTell™ Red 650

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 650 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™ Red 650 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™ Red 650 may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ Red has a peak excitation of 630 nm and can be excited by the red (633 nm) laser line. It has a peak emission of 660 nm and can be detected with a 660/20 band pass filter (equivalent to APC, Alexa Fluor® 647, or Cy5®), making it compatible with applications that utilize GFP or FITC antibodies for multicolor cell analysis.

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.

Spectrum

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)
CytoTell™ Red 590	562	587

Citations

View all 19 citations: Citation Explorer

Specific ECM degradation potentiates the antitumor activity of CAR-T cells in solid tumors
Authors: Zheng, Rui and Shen, Kuo and Liang, Sixin and Lyu, Yanhong and Zhang, Siyan and Dong, Hao and Li, Yuanfeng and Han, Yujie and Zhao, Xiaojuan and Zhang, Yiting and others,
Journal: Cellular \& Molecular Immunology (2024): 1--14

IL-21-and CXCL9-engineered GPC3-specific CAR-T cells combined with PD-1 blockade enhance cytotoxic activities against hepatocellular carcinoma
Authors: Chen, Shanshan and Gong, Fusheng and Liu, Shijia and Xie, Yunqing and Ye, Xingming and Lin, Xiaowei and Wang, Xiangru and Zheng, Qiuhong and Liu, Qinying and Sun, Yang
Journal: Clinical and Experimental Medicine (2024): 204

Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions
Authors: Kubota, Sho and Sun, Yuqi and Morii, Mariko and Bai, Jie and Ideue, Takako and Hirayama, Mayumi and Sorin, Supannika and Eerdunduleng, and Yokomizo-Nakano, Takako and Osato, Motomi and others,
Journal: The EMBO Journal (2024): 1--24

T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation
Authors: Katsuyama, Naoya and Kawase, Takakazu and Barakat, Carolyne and Mizuno, M and Tomita, Akihiro and Ozeki, Kazutaka and Nishio, Nobuhiro and Sato, Yoshie and Kajiya, Ryoko and Shiraishi, Keiko and others,
Journal: Nagoya J Med Sci (2023)

$\alpha$$\beta$-T-cell receptor transduction gives superior mitochondrial function to $\gamma$$\delta$-T cells with promising persistence
Authors: Ishihara, Mikiya and Miwa, Hiroshi and Fujiwara, Hiroshi and Akahori, Yasushi and Kato, Takuma and Tanaka, Yoshimasa and Tawara, Isao and Shiku, Hiroshi
Journal: iScience (2023)

Page updated on December 17, 2024

Ordering information

Price
Unit size	500 Tests 2x500 Tests
Catalog Number	2225522256
Quantity

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Additional ordering information

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Physical properties

Molecular weight	~600
Solvent	DMSO

Spectral properties

Excitation (nm)	626
Emission (nm)	643

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