CytoTell™ Green

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22253 $95


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)510/525
MW419.24
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Cell Biology
Labeling Cells
Related Fluorescence Imaging
Viability and Proliferation
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.




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.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Wavelength (nm)





Protocol


Quick Preview

This protocol only provides a guideline, and should be modified according to your specific needs.

1. Prepare 500 X DMSO stock solution

Add 500 µL DMSO into the dye powder vial, mix it well by vortexing to have a 500X DMSO stock solution

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.

 

2. Prepare 1X dye working solution

Prepare a 1X dye working solution by diluting 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 a t en fold range. Such as CytoTell™ Red might use much less amount in some cell types than the recommend concentrations.

 

3. Analyze cells with a flow cytometer or a fluorescence microscope:

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

 

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

 

3.3     Resuspend cells in 500 μL of the dye working solution (from Step 2).

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)

 

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

 

3.5     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.

 

3.6    Monitor the fluorescence change at respected Ex/Em (see Table 1) with a flow cytometer or a fluorescence microscope.






References

Effector T Cell Resistance to Suppression and STAT3 Signaling during the Development of Human Type 1 Diabetes
Authors: Emmi-Leena Ihantola, Tyyne Viisanen, Ahmad M Gazali, Kirsti Näntö-Salonen, Auni Juutilainen, Leena Moilanen, Reeta Rintamäki, Jussi Pihlajamäki, Riitta Veijola, Jorma Toppari
Journal: The Journal of Immunology (2018): ji1701199

Nanocomposite injectable gels capable of self-replenishing regenerative extracellular microenvironments for in vivo tissue engineering
Authors: Koji Nagahama, Naho Oyama, Kimika Ono, Atsushi Hotta, Keiko Kawauchi, Takahito Nishikata
Journal: Biomaterials Science (2018)

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

CXCL12--CXCR4 Axis Is Required for Contact-Mediated Human B Lymphoid and Plasmacytoid Dendritic Cell Differentiation but Not T Lymphoid Generation
Authors: Hirohito Minami, Keiki Nagaharu, Yoshiki Nakamori, Kohshi Ohishi, Naoshi Shimojo, Yuki Kageyama, Takeshi Matsumoto, Yuka Sugimoto, Isao Tawara, Masahiro Masuya
Journal: The Journal of Immunology (2017): ji1700054

Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells
Authors: Volker Klöss, Oliver Grünvogel, Guido Wabnitz, Tatjana Eigenbrod, Stefanie Ehrhardt, Felix Lasitschka, Volker Lohmann, Alexander H Dalpke
Journal: Frontiers in Immunology (2017): 1238

Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages
Authors: Junko Tsuboki, Yukio Fujiwara, Hasita Horlad, Daisuke Shiraishi, Toshihiro Nohara, Shingo Tayama, Takeshi Motohara, Yoichi Saito, Tsuyoshi Ikeda, Kiyomi Takaishi
Journal: Scientific Reports (2016)

Multiplexing analysis of cell proliferation and cellular functions using a new multicolor panel of fluorescent cell proliferation dyes (P1290)
Authors: Jinfang Liao, Qin Zhao, Yibo Wu, Zhenjun Diwu
Journal: The Journal of Immunology (2013): 119--4

JSRM Code: 013020300010 Full Text PDF
Authors: A Bertolo, D Pavlicek, A Gemperli, M Baur, T Pötzel, J Stoyanov
Journal: Unknown