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CytoTell™ Red 590

Cell tracking assay using CytoTell™ Red 590. Jurkat cells (~2x10^6 cells/mL) were stained with CytoTell™ Red 590 on day 0. Cells were passed serially at 1:1 ratio for 7 days. Fluorescence intensity was measured using FACS Calibur flow cytometer in FL2 channel. Successive generations were represented by different colors.
Cell tracking assay using CytoTell™ Red 590. Jurkat cells (~2x10^6 cells/mL) were stained with CytoTell™ Red 590 on day 0. Cells were passed serially at 1:1 ratio for 7 days. Fluorescence intensity was measured using FACS Calibur flow cytometer in FL2 channel. Successive generations were represented by different colors.
Ordering information
Price ()
Catalog Number22261
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 weight619.53
SolventDMSO
Spectral properties
Excitation (nm)562
Emission (nm)587
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
619.53
Excitation (nm)
562
Emission (nm)
587
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™ Red 590 is a red 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. Up to 8 generations may be visualized. CytoTell™ Red 590 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™ Red 590 may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ Red 590 has a peak excitation of 570 nm and can be excited by the yellow (561 nm) laser line. It has a peak emission of 590 nm and can be detected with a 610/20 band pass filter, making it compatible with applications that utilize GFP or FITC antibodies for multicolor cell analysis.

Platform


Flow cytometer

Excitation488 nm or 561nm laser
Emission610/20 nm filter
Instrument specification(s)PE-Texas Red 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™ Red 590 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 mM161.413 µL807.063 µL1.614 mL8.071 mL16.141 mL
5 mM32.283 µL161.413 µL322.825 µL1.614 mL3.228 mL
10 mM16.141 µL80.706 µL161.413 µL807.063 µL1.614 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)562
Emission (nm)587

Citations


View all 7 citations: Citation Explorer
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
Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells
Authors: Klöss, Volker and Grünvogel, Oliver and Wabnitz, Guido and Eigenbrod, Tatjana and Ehrhardt, Stefanie and Lasitschka, Felix and Lohmann, Volker and Dalpke, Alex and er H, undefined
Journal: Frontiers in Immunology (2017): 1238
CXCL12--CXCR4 Axis Is Required for Contact-Mediated Human B Lymphoid and Plasmacytoid Dendritic Cell Differentiation but Not T Lymphoid Generation
Authors: Minami, Hirohito and Nagaharu, Keiki and Nakamori, Yoshiki and Ohishi, Kohshi and Shimojo, Naoshi and Kageyama, Yuki and Matsumoto, Takeshi and Sugimoto, Yuka and Tawara, Isao and Masuya, Masahiro and others, undefined
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: Kl{\"o}ss, Volker and Gr{\"u}nvogel, Oliver and Wabnitz, Guido and Eigenbrod, Tatjana and Ehrhardt, Stefanie and Lasitschka, Felix and Lohmann, Volker and Dalpke, Alexander H
Journal: Frontiers in immunology (2017): 1238
Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages
Authors: Tsuboki, Junko and Fujiwara, Yukio and Horlad, Hasita and Shiraishi, Daisuke and Nohara, Toshihiro and Tayama, Shingo and Motohara, Takeshi and Saito, Yoichi and Ikeda, Tsuyoshi and Takaishi, Kiyomi and others, undefined
Journal: Scientific Reports (2016)
Multiplexing analysis of cell proliferation and cellular functions using a new multicolor panel of fluorescent cell proliferation dyes (P1290)
Authors: Liao, Jinfang and Zhao, Qin and Wu, Yibo and Diwu, Zhenjun
Journal: The Journal of Immunology (2013): 119--4

Application notes


Annexin V