AAT Bioquest

CytoCalcein™ Violet 660, AM *Excited at 405 nm*

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Physical properties
Molecular weight812.87
Spectral properties
Excitation (nm)402
Emission (nm)660
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
CytoCalcein™ Violet 660 AM ester is designed for labeling live cells in the same way as Calcein, AM does. It is well excited with the Violet laser at 405 nm, which is now equipped in most flow cytometers. It is also well-excited by the excitation sources of fluorescence microscopes. Upon getting into live cells the CytoCalcein™ Violet 660 is hydrolyzed into a fluorescent dye of multiple negative charges, which makes the dye well retained in live cells. It has the largest Stokes Shift among the known live cell stains. This exceptional spectral separation from the typical existing FACS fluorophores provides additional options for multiplexing experiments with a flow cytometer or a fluorescence microscope. CytoCalcein™ Violet 660 has been developed for multiplexing flow cytometric and fluorescence imaging applications. It provides a new unique color for flow cytometric analysis of live cells.


Flow cytometer

Excitation405 nm laser
Emission660/20 nm filter

Fluorescence microscope

Excitation405 nm laser
Emission675/30 nm
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Recommended plateSolid black

Example protocol


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

CytoCalcein™ Violet 660 Stock Solution
  1. Prepare a 2 to 5 mM stock solution of CytoCalcein™ Violet 660 in high-quality, anhydrous DMSO.

    Note: The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.


CytoCalcein™ Violet 660 Working Solution
  1. Prepare a CytoCalcein™ Violet 660 working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, CytoCalcein™ Violet 660 at the final concentration of 4 to 5 µM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.

    Note: If your cells contain organic anion transporters, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the working solution to reduce leakage of the de-esterified indicators.


  1. Prepare cells for imaging.

  2. Remove the cell culture medium and wash cells once with serum-free buffer to remove any remaining media.

    Note: Serum in cell culture media may contain esterase activity, which can increase background interference.

  3. Add CytoCalcein™ Violet 660 working solution to the culture.

  4. Incubate cells at 37 °C for 30 to 60 minutes.

  5. Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.

  6. Measure the fluorescence intensity using either a fluorescence microscope equipped with a violet laser and 675/30 nm filter set, a flow cytometer equipped with a violet laser and a 660/20 nm filter, or a fluorescence plate reader at Ex/Em = 405/660 nm cutoff 630 nm.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of CytoCalcein™ Violet 660, AM *Excited at 405 nm* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM123.021 µL615.105 µL1.23 mL6.151 mL12.302 mL
5 mM24.604 µL123.021 µL246.042 µL1.23 mL2.46 mL
10 mM12.302 µL61.51 µL123.021 µL615.105 µL1.23 mL

Molarity calculator

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

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Spectral properties

Excitation (nm)402
Emission (nm)660


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