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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Ex/Em (nm)409/443
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.

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 =

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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.


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