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CytoTell™ Red 650

Cell proliferation assay with CytoTell™Red 650. Jurkat cells are stained with  CytoTell™ Red 650 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 APC channel.
Cell proliferation assay with CytoTell™Red 650. Jurkat cells are stained with  CytoTell™ Red 650 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 APC channel.
Effect of ONA on tumour cell proliferation in co-culture of EOC cells with HMDMs. Compared with EOC mono-culture (A-i), non-treated (A-ii) and onionin A (ONA) (10 μM)-treated HMDMs (A-iii) were incubated with epithelial ovarian cancer (EOC) cells (SKOV3, ES2, and RMG1) for 24 hours, followed by the determination of BrdU-positive cells by ELISA (B). Compared with EOC mono-culture (C-i) and co-culture without ONA treatment (C-ii), co- cultured cells (HMDMs and EOC cells) were treated with the indicated concentrations of ONA (3–10 μM) for 24 hours (C-iii), followed by the determination of BrdU-positive cells by ELISA (D). SKOV3 cells were labelled with CytoTell Red fluorescence and co-cultured with HMDMs, and then cell proliferation was assessed by flow cytometry (E). IL-10 production was evaluated by ELISA (F). HMDM-derived soluble factors were evaluated by a cytokine array kit, and the relative spot densities of cystatin C, macrophage migration inhibitory factor (MIF), growth differentiation factor (GDF)-15, urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 9 (MMP9), epidermal growth factor (EGF), and IL-1 receptor antagonist (IL-1Ra) were evaluated using the ImageJ software programme (G). The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01 vs. control. Source: <strong>Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages </strong>by Tsuboki et al., <em>Scientific Reports</em>,  July 2016.
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Price ()
Catalog Number22255
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight600
SolventDMSO
Spectral properties
Excitation (nm)626
Emission (nm)643
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
600
Excitation (nm)
626
Emission (nm)
643
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 650 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 650 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 650 may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ Red has a peak excitation of 630 nm and can be excited by the red (633 nm) laser line. It has a peak emission of 660 nm and can be detected with a 660/20 band pass filter (equivalent to APC, Alexa Fluor® 647, or Cy5®), making it compatible with applications that utilize GFP or FITC antibodies for multicolor cell analysis.

Platform


Flow cytometer

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC 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 650 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 mM166.667 µL833.333 µL1.667 mL8.333 mL16.667 mL
5 mM33.333 µL166.667 µL333.333 µL1.667 mL3.333 mL
10 mM16.667 µL83.333 µL166.667 µL833.333 µL1.667 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)626
Emission (nm)643

Citations


View all 11 citations: Citation Explorer
Combination therapy with B7H3-redirected bispecific antibody and Sorafenib elicits enhanced synergistic antitumor efficacy
Authors: Huang, Cheng and Li, Hongjian and Feng, Yunyu and Li, Xiaoling and Zhang, Zongliang and Jiang, Caiying and Wang, Jichao and Yang, Chenli and Fu, Yuying and Mu, Min and others,
Journal: Theranostics (2020): 10498
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
Metabolic Switching of Tumor Cells under Hypoxic Conditions in a Tumor-on-a-chip Model
Authors: Palacio-Casta{\~n}eda, Valentina and Kooijman, Lucas and Venzac, Bastien and Verdurmen, Wouter PR and Le Gac, S{\'e}verine
Journal: Micromachines (2020): 382
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)

Application notes


Annexin V