CytoTell™ Red 650

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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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

Overview SDS Protocol

Molecular weight

~600

Excitation (nm)

626

Emission (nm)

643

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.

Platform

Flow cytometer

Excitation	640 nm laser
Emission	660/20 nm filter
Instrument specification(s)	APC 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.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	626
Emission (nm)	643

Product Family

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

Images

Figure 1. Cell proliferation assay with CytoTell™Red 650. Jurkat cells are stained with CytoTell™ Red 650 on Day0, and serially passed at 1:1 ratio for 8 days. Fluorescence intensity of each generation was measured with ACEA NovoCyte 3000 flow cytometer APC channel.

Figure 2. Effect of ONA on tumour cell proliferation in co-culture of EOC cells with HMDMs. Compared with EOC mono-culture (A-i), non-treated (A-ii) and onionin A (ONA) (10 μM)-treated HMDMs (A-iii) were incubated with epithelial ovarian cancer (EOC) cells (SKOV3, ES2, and RMG1) for 24 hours, followed by the determination of BrdU-positive cells by ELISA (B). Compared with EOC mono-culture (C-i) and co-culture without ONA treatment (C-ii), co- cultured cells (HMDMs and EOC cells) were treated with the indicated concentrations of ONA (3–10 μM) for 24 hours (C-iii), followed by the determination of BrdU-positive cells by ELISA (D). SKOV3 cells were labelled with CytoTell Red fluorescence and co-cultured with HMDMs, and then cell proliferation was assessed by flow cytometry (E). IL-10 production was evaluated by ELISA (F). HMDM-derived soluble factors were evaluated by a cytokine array kit, and the relative spot densities of cystatin C, macrophage migration inhibitory factor (MIF), growth differentiation factor (GDF)-15, urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 9 (MMP9), epidermal growth factor (EGF), and IL-1 receptor antagonist (IL-1Ra) were evaluated using the ImageJ software programme (G). The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01 vs. control. Source: Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages by Tsuboki et al., Scientific Reports, July 2016.

Citations

View all 15 citations: Citation Explorer

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)

Development of TCR-T cell therapy targeting mismatched HLA-DPB1 for relapsed leukemia after allogeneic transplantation
Authors: Barakat, Carolyne and Inagaki, Yuichiro and Mizuno, Shohei and Nishio, Nobuhiro and Katsuyama, Naoya and Sato, Yoshie and Kobayashi, Miki and Ozeki, Kazutaka and Iida, Hiroatsu and Tomita, Akihiro and others,
Journal: International Journal of Hematology (2023): 1--15

$\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)

Combination therapy with B7H3-redirected bispecific antibody and Sorafenib elicits enhanced synergistic antitumor efficacy
Authors: Huang, Cheng and Li, Hongjian and Feng, Yunyu and Li, Xiaoling and Zhang, Zongliang and Jiang, Caiying and Wang, Jichao and Yang, Chenli and Fu, Yuying and Mu, Min and others,
Journal: Theranostics (2020): 10498

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells
Authors: Ozay, E Ilker and Shanthalingam, Sudarvili and Sherman, Heather L and Torres, Joe A and Osborne, Barbara A and Tew, Gregory N and Minter, Lisa M
Journal: Molecular Therapy (2020): 1987--2006

Metabolic Switching of Tumor Cells under Hypoxic Conditions in a Tumor-on-a-chip Model
Authors: Palacio-Casta{\~n}eda, Valentina and Kooijman, Lucas and Venzac, Bastien and Verdurmen, Wouter PR and Le Gac, S{\'e}verine
Journal: Micromachines (2020): 382

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

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

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

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