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CytoTell™ Green

Cell proliferation assay with CytoTell™ Green and CFSE.  Jurkat cells (~2×10<sup>6</sup> cells/mL) were stained with CytoTell™ Green or CFSE 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 in FITC channel on the day of passage. Successive generations were represented by different colors.
Cell proliferation assay with CytoTell™ Green and CFSE.  Jurkat cells (~2×10<sup>6</sup> cells/mL) were stained with CytoTell™ Green or CFSE 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 in FITC channel on the day of passage. Successive generations were represented by different colors.
Ordering information
Price ()
Catalog Number22253
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight419.24
SolventDMSO
Spectral properties
Excitation (nm)510
Emission (nm)525
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
419.24
Excitation (nm)
510
Emission (nm)
525
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, there are a few severe problems associated with the use of CFSE for monitoring cell proliferation. 1). CFSE is highly toxic to cells. CFSE indiscriminately reacts with all amino groups, thus changes many critical intracellular protein functions (such as cell membrane GPCRs); 2). CFSE has slow response and is inconvenient to use. The CFSE fluorescence intensity of the 2nd generation cells is decreased more than 10 fold from the 1st generation. You would have to wait for another generation to start the cell proliferation analysis. 3). Medium removal is required. You would have to remove medium for cell analysis with a flow cytometer since CFSE reacts with medium components. CytoTell™ Green is developed to eliminate these limitations. It has distinct advantages over CFSE. 1). CytoTell™ Green has very minimal cytotoxicity compared to CFSE. It is well retained in cells since the probe is designed to minimize the MDR interaction that generally pumps many cell stains out cells; 2). CytoTell™ Green exhibits much faster response and is more convenient to use than CFSE. There is no fluorescence intensity gap between 1st and 2nd generation of cells. As cells divide, CytoTell™ Green is distributed equally between daughter cells that can be measured as successive halving of the fluorescence intensity of the dye; 3). There is no need to remove medium since CytoTell™ Green does not react with medium components; 4). CytoTell™ Green is more sensitive than CFSE. Up to 9 generations may be visualized; 5). CytoTell™ Green is much more stable than CFSE. CytoTell™ Green stock solution can be stored at room temperature for a few days. CytoTell™ Green 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™ Green may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ Green has a peak excitation of 520 nm and can be excited by the blue (488 nm) laser line, making it compatible with FITC filter set.

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds
  2. Add 1X dye working solution
  3. Incubate dyes with cells at room temperature or 37 oC for 10 to 30 minutes
  4. Remove the dye working solution
  5. 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

CytoTellTM 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

  1. Treat cells with test compounds for a desired period of time.

  2. Centrifuge the cells to get 1-5 × 105 cells per tube.

  3. 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)

  4. Incubate cells with a dye solution at room temperature or 37 °C for 10 to 30 minutes, protected from light.

  5. 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.

  6. 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™ Green to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM238.527 µL1.193 mL2.385 mL11.926 mL23.853 mL
5 mM47.705 µL238.527 µL477.054 µL2.385 mL4.771 mL
10 mM23.853 µL119.263 µL238.527 µL1.193 mL2.385 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


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spectrum

Spectral properties

Excitation (nm)510
Emission (nm)525

Citations


View all 21 citations: Citation Explorer
Concurrent stimulation of monocytes with CSF1 and polarizing cytokines reveals phenotypic and functional differences with classical polarized macrophages
Authors: An, Liying and Michaeli, Julia and Pallavi, Prama and Breedijk, Annette and Xu, Xin and Dietrich, Nadine and Sigl, Martin and Keese, Michael and Nitschke, Katja and Jarczyk, Jonas and others,
Journal: Journal of Leukocyte Biology (2022)
STAT3 governs the HIF-1$\alpha$ response in IL-15 primed human NK cells
Authors: Coulibaly, Anna and Vel{\'a}squez, Sonia Y and Kassner, Nina and Schulte, Jutta and Barbarossa, Maria Vittoria and Lindner, Holger A
Journal: Scientific reports (2021): 1--13
Selective involution of thymic medulla by cyclosporine A with a decrease of mature thymic epithelia, XCR1+ dendritic cells, and epithelium-free areas containing Foxp3+ thymic regulatory T cells
Authors: Sawanobori, Yasushi and Kitazawa, Yusuke and Ueta, Hisashi and Matsuno, Kenjiro and Tokuda, Nobuko
Journal: Histochemistry and Cell Biology (2021): 1--14
T-cell dysfunction and autoantigen recognition in type 1 diabetes
Authors: Ihantola, Emmi-Leena
Journal: (2021)
Characterization of proinsulin T cell epitopes restricted by type 1 diabetes--associated HLA class II molecules
Authors: Ihantola, Emmi-Leena and Ilmonen, Henna and Kailaanm{\"a}ki, Anssi and Rytk{\"o}nen-Nissinen, Marja and Azam, Aur{\'e}lien and Maill{\`e}re, Bernard and Arlehamn, Cecilia S Lindestam and Sette, Alessandro and Motwani, Keshav and Seay, Howard R and others,
Journal: The Journal of Immunology (2020): 2349--2359
N-Octanoyl-Dopamine inhibits cytokine production in activated T-cells and diminishes MHC-class-II expression as well as adhesion molecules in IFNγ-stimulated endothelial cells
Authors: Hofmann, Björn B and Krapp, Nicolas and Li, Yingchun and De La Torre, Carolina and Sol, Marloes and Braun, Jana D and Kolibabka, Matthias and Pallavi, Prama and Krämer, Bernhard K and Yard, Benito A and others, undefined
Journal: Scientific Reports (2019): 1--11
N-Octanoyl-Dopamine inhibits cytokine production in activated T-cells and diminishes MHC-class-II expression as well as adhesion molecules in IFN$\gamma$-stimulated endothelial cells
Authors: Hofmann, Bj{\"o}rn B and Krapp, Nicolas and Li, Yingchun and De La Torre, Carolina and Sol, Marloes and Braun, Jana D and Kolibabka, Matthias and Pallavi, Prama and Kr{\"a}mer, Bernhard K and Yard, Benito A and others,
Journal: Scientific reports (2019): 1--11
Early KLRG1+ but Not CD57+ CD8+ T Cells in Primary Cytomegalovirus Infection Predict Effector Function and Viral Control
Authors: Hoji, Aki and Popescu, Iulia D and Pipeling, Matthew R and Shah, Pali D and Winters, Spencer A and McDyer, John F
Journal: The Journal of Immunology (2019): 2063--2075
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