CytoTell™ Blue

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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.
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Unit Size: Cat No: Price (USD): Qty:
22251 $95


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

Ex/Em (nm)409/443
MW379.71
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, 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.




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.



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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Move mouse over grid to display wavelength & intensity values.

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Wavelength (nm)





Protocol


Quick Preview

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

Key parameters
Instrument:Flow cytometer
Excitation:Pacific Blue filter set
Emission:Pacific Blue filter set
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.
Example data analysis and figures

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.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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References

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