logo
AAT Bioquest

CytoTell™ Red 650

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.

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.

Spectrum

Product family

NameExcitation (nm)Emission (nm)
CytoTell™ Red 590562587

Citations

View all 18 citations: Citation Explorer
IL-21-and CXCL9-engineered GPC3-specific CAR-T cells combined with PD-1 blockade enhance cytotoxic activities against hepatocellular carcinoma
Authors: Chen, Shanshan and Gong, Fusheng and Liu, Shijia and Xie, Yunqing and Ye, Xingming and Lin, Xiaowei and Wang, Xiangru and Zheng, Qiuhong and Liu, Qinying and Sun, Yang
Journal: Clinical and Experimental Medicine (2024): 204
Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions
Authors: Kubota, Sho and Sun, Yuqi and Morii, Mariko and Bai, Jie and Ideue, Takako and Hirayama, Mayumi and Sorin, Supannika and Eerdunduleng, and Yokomizo-Nakano, Takako and Osato, Motomi and others,
Journal: The EMBO Journal (2024): 1--24
T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation
Authors: Katsuyama, Naoya and Kawase, Takakazu and Barakat, Carolyne and Mizuno, M and Tomita, Akihiro and Ozeki, Kazutaka and Nishio, Nobuhiro and Sato, Yoshie and Kajiya, Ryoko and Shiraishi, Keiko and others,
Journal: Nagoya J Med Sci (2023)
$\alpha$$\beta$-T-cell receptor transduction gives superior mitochondrial function to $\gamma$$\delta$-T cells with promising persistence
Authors: Ishihara, Mikiya and Miwa, Hiroshi and Fujiwara, Hiroshi and Akahori, Yasushi and Kato, Takuma and Tanaka, Yoshimasa and Tawara, Isao and Shiku, Hiroshi
Journal: iScience (2023)
Development of TCR-T cell therapy targeting mismatched HLA-DPB1 for relapsed leukemia after allogeneic transplantation
Authors: Barakat, Carolyne and Inagaki, Yuichiro and Mizuno, Shohei and Nishio, Nobuhiro and Katsuyama, Naoya and Sato, Yoshie and Kobayashi, Miki and Ozeki, Kazutaka and Iida, Hiroatsu and Tomita, Akihiro and others,
Journal: International Journal of Hematology (2023): 1--15
Page updated on October 11, 2024

Ordering information

Price
Unit size
500 Tests
2x500 Tests
Catalog Number
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

~600

Solvent

DMSO

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Flow cytometer

Excitation640 nm laser
Emission660, 20 nm filter
Instrument specification(s)APC channel
Cell proliferation assay with CytoTell&trade;Red 650. Jurkat cells are stained with&nbsp; CytoTell&trade;&nbsp;Red&nbsp;650&nbsp;on Day0, and serially passed at 1:1 ratio for 8 days. Fluorescence intensity of each generation was measured with ACEA&nbsp; NovoCyte 3000 flow cytometer APC channel.
Cell proliferation assay with CytoTell&trade;Red 650. Jurkat cells are stained with&nbsp; CytoTell&trade;&nbsp;Red&nbsp;650&nbsp;on Day0, and serially passed at 1:1 ratio for 8 days. Fluorescence intensity of each generation was measured with ACEA&nbsp; NovoCyte 3000 flow cytometer APC channel.
Cell proliferation assay with CytoTell&trade;Red 650. Jurkat cells are stained with&nbsp; CytoTell&trade;&nbsp;Red&nbsp;650&nbsp;on Day0, and serially passed at 1:1 ratio for 8 days. Fluorescence intensity of each generation was measured with ACEA&nbsp; 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&thinsp;&mu;M)-treated HMDMs (A-iii) were incubated with epithelial ovarian cancer (EOC) cells (SKOV3, ES2, and RMG1) for 24&thinsp;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&ndash;10&thinsp;&mu;M) for 24&thinsp;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&thinsp;&plusmn;&thinsp;SD. *p-value&thinsp;&lt;&thinsp;0.05, **p-value&thinsp;&lt;&thinsp;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>, &nbsp;July 2016.