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Enabling Multicolor Cell Proliferation Assay Panel
CytoTell® Green
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, these include:
- CFSE is highly toxic to cells since CFSE indiscriminately reacts with all amino groups, thus affects many critical intracellular protein functions (such as cell membrane GPCRs).
- 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.
- Medium removal is required. You would have to remove medium for cell analysis with a flow cytometer since CFSE reacts with medium components.
Fig. 1
CytoTell™ dye working principle. CytoTell™ dye consists of three components: a) fluorescence blocker; b) masked fuorephore; and c) cell-retaining moiety. Upon entering live cells, the fluorescence of CytoTell™ dye is released via the removal of fluorescence blocker, and the released fluorephore is retained in cells through the cell-retaining group.
Fig. 2
Emission spectral comparison of CytoTell™ Blue (Ex/Em = 403/454 nm), CytoTell™ Green (Ex/Em = 511/525 nm), and CytoTell™ Red (Ex/Em = 628/643 nm) in PBS buffer (pH 7.2).
CytoTell® Green is developed to eliminate the above CFSE limitations. CytoTell® Green 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® Green may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers. CytoTell® Green can be excited by the 488 nm blue laser line with the peak emission at 520 nm, which makes it compatible with the FITC filter set.
Fig. 3
Left: Cell tracking assay with CytoTell™ Green and CFSE. Jurkat cells (~2x106 cells/mL) were stained with CytoTell™ Green or CFSE (0.5 µM) on Day 0. The cells were passed serially at 1:1 ratio for 9 days. Fluorescence intensity was measured with FACS Calibur flow cytometer (BD, San Jose, CA) in FL1 channel on the day after passage. Successive generations were represented by different colors. Right: Stability comparison of CytoTell™ Green and CFSE. 5 mM PBS working solutions of CytoTell™ Green and CFSE were monitored using HPLC (pH 7.2).
Key Features of CytoTell® Green:
- Spectrally similar to CFSE and FITC.
- Much faster response to cell proliferation than CFSE.
- More convenient to use than CFSE.
- More sensitive than CFSE.
- Much more stable than CFSE.
CytoTell® Blue
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 those of GFP or FITC. CytoTell® dyes are well excited with major laser lines such as 405 nm, 488 nm or 633 nm laser line with multicolor emissions. They 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 those of fluorescein. CytoTell® Blue is a blue fluorescent dye that stains cells evenly. It has a peak excitation of 405 nm and can be excited by the 405 nm violet laser line. Its peak emission of 450 nm can be detected with a 450/20 band pass filter (equivalent to Pacific Blue® or BD Horizon® V450), making it compatible with applications that use GFP or FITC antibodies for multicolor cell analysis.
CytoTell® Red
Fig. 4
Left: The excitation and emission spectra of CytoTell™ Red in PBS buffer (pH 7.2). Right: Cell tracking assay with CytoTell™ Red. Jurkat cells (~ 2x106 cells/mL) were stained with CytoTell™ Red (2 µM) on Day 0. The cells were passed serially at 1:1 ratio for 11 days. Fluorescence intensity was measured with FACS Calibur flow cytometer (BD, San Jose, CA) in FL4 channel on the day after passage. Successive generations were represented by different colors.
Document: 03.0012.121001r1
Last updated Fri Oct 10 2025
Enabling Multicolor Cell Proliferation Assay Panel