CytoTell™ UltraGreen

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Cell tracking assay using CytoTell™ UltraGreen. Jurkat cells (~2x10^6 cells/mL) were stained with CytoTell™ UltraGreen on Day 0. Cells were passed serially at 1:1 ratio for 7 days. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in FITC channel. Successive generations were represented by different colors.
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Storage Freeze (<-15 °C)
Minimize light exposure
Category Cell Biology
Labeling Cells
Related Fluorescence Imaging
Viability and Proliferation
Secondary Reagents
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, there are a few severe problems associated with the use of CFSE for monitoring cell proliferation. 1). CFSE is highly toxic to cells. CFSE indiscriminately reacts with all amino groups, thus changes many critical intracellular protein functions (such as cell membrane GPCRs); 2). CFSE has slow response and is inconvenient to use. The CFSE fluorescence intensity of the 2nd generation cells is decreased more than 10 fold from the 1st generation. You would have to wait for another generation to start the cell proliferation analysis. 3). Medium removal is required. You would have to remove medium for cell analysis with a flow cytometer since CFSE reacts with medium components. CytoTell™ Green has been developed to eliminate these CFSE limitations. Based on our customers' feedbacks on our CytoTell™ Green, CytoTell™ UltraGreen is our newest improvement. It has distinct advantages. 1). CytoTell™ UltraGreen is well retained in cells; 2). CytoTell™ UltraGreen exhibits much faster response and is more convenient to use than CFSE. The fluorescence intensity gap between 1st and 2nd generation is significantly minimized. As cells divide, CytoTell™ UltraGreen is distributed equally between daughter cells that can be measured as successive halving of the fluorescence intensity of the dye; 3). CytoTell™ UltraGreen is more sensitive than CFSE. Up to 9 generations may be visualized; 4). CytoTell™ UltraGreen is much more stable than CFSE. CytoTell™ UltraGreen stock solution can be stored at room temperature for a few days. CytoTell™ UltraGreen 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™ UltraGreen may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell™ UltraGreen has a peak excitation of 519 nm and can be excited by the blue (488 nm) laser line, making it compatible with FITC filter set.

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of CytoTell™ UltraGreen 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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