CytoTell™ Blue

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	379.71
Solvent	DMSO

Spectral properties

Excitation (nm)	410
Emission (nm)	445

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

Alternative formats

Overview SDS Protocol

Molecular weight

379.71

Excitation (nm)

410

Emission (nm)

445

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™ Blue is a blue 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™ Blue may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ Blue has a peak excitation of 405 nm and can be excited by the violet (405 nm) laser line. It has a peak emission of 450 nm and can be detected with a 450/20 band pass filter (equivalent to Pacific Blue®, or BD Horizon® V450), making it compatible with applications that utilize GFP or FITC antibodies for multicolor cell analysis.

Platform

Flow cytometer

Excitation	405 nm laser
Emission	450/40 nm filter
Instrument specification(s)	Pacific Blue 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™ Blue 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	263.359 µL	1.317 mL	2.634 mL	13.168 mL	26.336 mL
5 mM	52.672 µL	263.359 µL	526.718 µL	2.634 mL	5.267 mL
10 mM	26.336 µL	131.679 µL	263.359 µL	1.317 mL	2.634 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

Spectral properties

Excitation (nm)	410
Emission (nm)	445

Product Family

Name	Excitation (nm)	Emission (nm)
CytoTell™ Green	510	525
CytoTell™ Orange	541	560

Images

Figure 1. Cell proliferation assay with CytoTell™Blue. Jurkat cells are stained with CytoTell™ Blue 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 Pacific Blue channel.

Citations

View all 16 citations: Citation Explorer

Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques
Authors: Rahman, MA and Bissa, M and Silva de Castro, I and Helmold Hait, S and Stamos, JD and Bhuyan, F and Hunegnaw, R and Sarkis, S and Gutowska, A and Doster, MN and others,
Journal: Nature Microbiology (2023): 1--14

The impact of CCR8+ regulatory T cells on cytotoxic T cell function in human lung cancer
Authors: Haruna, Miya and Ueyama, Azumi and Yamamoto, Yoko and Hirata, Michinari and Goto, Kumiko and Yoshida, Hiroshi and Higuchi, Naoko and Yoshida, Tetsuya and Kidani, Yujiro and Nakamura, Yamami and others,
Journal: Scientific Reports (2022): 1--12

Enhance anti-lung tumor efficacy of chimeric antigen receptor-T cells by ectopic expression of CC motif chemokine receptor 6
Authors: Jin, Liyuan and Cao, Lei and Zhu, Yingjie and Cao, Jiani and Li, Xiaoyan and Zhou, Jianxia and Liu, Bing and Zhao, Tongbiao
Journal: Science Bulletin (2020)

Interleukin-7 loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma
Authors: Huang, Jianhan and Zheng, Meijun and Zhang, Zongliang and Tang, Xin and Chen, Yaxing and Tong, Aiping and Zhou, Liangxue
Journal: (2020)

PD-1+ Tim3+ tumor-infiltrating CD8 T cells sustain the potential for IFN-$\gamma$ production, but lose cytotoxic activity in ovarian cancer
Authors: Sawada, Masaaki and Goto, Kumiko and Morimoto-Okazawa, Akiko and Haruna, Miya and Yamamoto, Kei and Yamamoto, Yoko and Nakagawa, Satoshi and Hiramatsu, Kosuke and Matsuzaki, Shinya and Kobayashi, Eiji and others,
Journal: International Immunology (2020): 397--405

Omega-3 docosahexaenoic acid (DHA) impedes silica-induced macrophage corpse accumulation by attenuating cell death and potentiating efferocytosis
Authors: Rajasinghe, Lichchavi D and Chauhan, Preeti S and Wierenga, Kathryn A and Evered, Augustus O and Harris, Shamya N and Bates, Melissa A and Gavrilin, Mikhail A and Pestka, James J
Journal: Frontiers in immunology (2020): 2179

Memo1-mediated tiling of radial glial cells facilitates cerebral cortical development
Authors: Nakagawa, Naoki and Plestant, Charlotte and Yabuno-Nakagawa, Keiko and Li, Jingjun and Lee, Janice and Huang, Chu-Wei and Lee, Amelia and Krupa, Oleh and Adhikari, Aditi and Thompson, Suriya and others,
Journal: Neuron (2019): 836--852

Improved survival of chimeric antigen receptor-engineered T (CAR-T) and tumor-specific T cells caused by anti-programmed cell death protein 1 single-chain variable fragment-producing CAR-T cells
Authors: Nakajima, Masao and Sakoda, Yukimi and Adachi, Keishi and Nagano, Hiroaki and Tamada, Koji
Journal: Cancer science (2019): 3079--3088

Progenitor hyperpolarization regulates the sequential generation of neuronal subtypes in the developing neocortex
Authors: Vitali, Ilaria and Fi{\`e}vre, Sabine and Telley, Ludovic and Oberst, Polina and Bariselli, Sebastiano and Frangeul, Laura and Baumann, Natalia and McMahon, John J and Klingler, Esther and Bocchi, Riccardo and others,
Journal: Cell (2018): 1264--1276

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

References

View all 50 references: Citation Explorer

Phenotyping of circulating CD8(+) T cell subsets in human cutaneous leishmaniasis
Authors: Khamesipour A, Nateghi Rostami M, Tasbihi M, Miramin Mohammadi A, Shahrestani T, Sarrafnejad A, Sohrabi Y, Esk and ari SE, Keshavarz Valian H.
Journal: Microbes Infect (2012): 702

Evaluation of multitype mathematical models for CFSE-labeling experiment data
Authors: Miao H, Jin X, Perelson AS, Wu H.
Journal: Bull Math Biol (2012): 300

Repetitive transplantation of different cell types sequentially improves heart function after infarction
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Journal: J Cell Mol Med (2012): 1640

Identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (CFSE)
Authors: Deleyrolle LP, Rohaus MR, Fortin JM, Reynolds BA, Azari H.
Journal: J Vis Exp. (2012)

New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes
Authors: Quah BJ, Parish CR.
Journal: J Immunol Methods (2012): 1

Dendritic cells restrict the transformation of Histoplasma capsulatum conidia into yeasts
Authors: Newman SL, Lemen W, Smulian AG.
Journal: Med Mycol (2011): 356

Allospecific regulatory effects of sirolimus and tacrolimus in the human mixed lymphocyte reaction
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Comparison of quantitative performance of three fluorescence labels in CE/LIF analysis of aspartate and glutamate in brain microdialysate
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The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation
Authors: Quah BJ, Parish CR.
Journal: J Vis Exp. (2010)