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CytoTell™ Red 650

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22256 $145

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Ex/Em (nm)628/643
Storage F/D/L
Category Cell Biology
Labeling Cells
Related Fluorescence Imaging
Viability and Proliferation
Biochemical Assays
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.

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.

Molarity calculator

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

Mass Molecular weight Volume Concentration Moles
/ = x =

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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 & Citations

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