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CytoTell™ UltraGreen

Cell tracking assay using CytoTell™ UltraGreen. Jurkat cells (~2x10^6 cells/mL) were stained with CytoTell™ UltraGreen on Day 0. Cells were passed serially at 1:1 ratio for 7 days. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in FITC channel. Successive generations were represented by different colors.
Cell tracking assay using CytoTell™ UltraGreen. Jurkat cells (~2x10^6 cells/mL) were stained with CytoTell™ UltraGreen on Day 0. Cells were passed serially at 1:1 ratio for 7 days. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in FITC channel. Successive generations were represented by different colors.
Ordering information
Price ()
Catalog Number22240
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 weight500
SolventDMSO
Spectral properties
Excitation (nm)492
Emission (nm)514
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
500
Excitation (nm)
492
Emission (nm)
514
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 has been developed to eliminate these CFSE limitations. Based on our customers' feedbacks on our CytoTell™ Green, CytoTell™ UltraGreen is our newest improvement. It has distinct advantages. 1). CytoTell™ UltraGreen is well retained in cells; 2). CytoTell™ UltraGreen exhibits much faster response and is more convenient to use than CFSE. The fluorescence intensity gap between 1st and 2nd generation is significantly minimized. As cells divide, CytoTell™ UltraGreen is distributed equally between daughter cells that can be measured as successive halving of the fluorescence intensity of the dye; 3). CytoTell™ UltraGreen is more sensitive than CFSE. Up to 9 generations may be visualized; 4). CytoTell™ UltraGreen is much more stable than CFSE. CytoTell™ UltraGreen stock solution can be stored at room temperature for a few days. CytoTell™ UltraGreen 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. CytoTell™ UltraGreen has a peak excitation of 519 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      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.

PREPARATION OF STOCK SOLUTIONS

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™ UltraGreen 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 mM200 µL1 mL2 mL10 mL20 mL
5 mM40 µL200 µL400 µL2 mL4 mL
10 mM20 µL100 µL200 µL1 mL2 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)492
Emission (nm)514

Citations


View all 15 citations: Citation Explorer
Development of double-positive thymocytes at single-cell resolution
Authors: Li, Young and Li, Kun and Zhu, Lianbang and Li, Bin and Zong, Dandan and Cai, Pengfei and Jiang, Chen and Du, Pengcheng and Lin, Jun and Qu, Kun
Journal: Genome medicine (2021): 1--18
Effect of impaired T-cell receptor signaling on the gut microbiota and systemic autoimmunity
Authors: Shirakashi, Mirei and Maruya, Mikako and Hirota, Keiji and Tsuruyama, Tatsuaki and Matsuo, Takashi and Watanabe, Ryu and Murata, Koichi and Tanaka, Masao and Ito, Hiromu and Yoshifuji, Hajime and others,
Journal: Arthritis \& Rheumatology (2021)
Conversion from epithelial to partial-EMT phenotype by Fusobacterium nucleatum infection promotes invasion of oral cancer cells
Authors: Shao, Wenhua and Fujiwara, Natsumi and Mouri, Yasuhiro and Kisoda, Satoru and Yoshida, Kayo and Yoshida, Kaya and Yumoto, Hiromichi and Ozaki, Kazumi and Ishimaru, Naozumi and Kudo, Yasusei
Journal: Scientific Reports (2021): 1--14
Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury
Authors: Yoshida, Kayo and Yoshida, Kaya and Fujiwara, Natsumi and Seyama, Mariko and Ono, Kisho and Kawai, Hotaka and Guo, Jiajie and Wang, Ziyi and Weng, Yao and Yu, Yaqiong and others,
Journal: Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease (2021): 166236
Newly emerged immunogenic neoantigens in established tumors enable hosts to regain immunosurveillance in a T-cell-dependent manner
Authors: Muramatsu, Tomoaki and Noguchi, Takuro and Sugiyama, Daisuke and Kanada, Yoshie and Fujimaki, Kaori and Ito, Sachiko and Gotoh, Momokazu and Nishikawa, Hiroyoshi
Journal: International Immunology (2021): 39--48
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
Evaluation of alloreactive T cells based on the degree of MHC incompatibility using flow cytometric mixed lymphocyte reaction assay in dogs
Authors: Miyamae, Jiro and Yagi, Hayato and Sato, Keita and Okano, Masaharu and Nishiya, Kohei and Katakura, Fumihiko and Sakai, Manabu and Nakayama, Tomohiro and Moritomo, Tadaaki and Shiina, Takashi
Journal: Immunogenetics (2019): 635--645
Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells
Authors: He, Xiaocui and Kl{\"a}sener, Kathrin and Iype, Joseena M and Becker, Martin and Maity, Palash C and Cavallari, Marco and Nielsen, Peter J and Yang, Jianying and Reth, Michael
Journal: The EMBO journal (2018)