AAT Bioquest

CytoCalcein™ Violet 500 *Excited at 405 nm*

Fluorescence image of HeLa cells stained with CytoCalcein™ Violet 500 *Excited at 405 nm* in a Costar black wall/clear bottom 96-well plate.
Fluorescence image of HeLa cells stained with CytoCalcein™ Violet 500 *Excited at 405 nm* in a Costar black wall/clear bottom 96-well plate.
Fluorescence image of HeLa cells stained with CytoCalcein™ Violet 500 *Excited at 405 nm* in a Costar black wall/clear bottom 96-well plate.
Images of Live HeLa cells stained with CytoCalcein Violet 500 (Cat.22013). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
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Physical properties
Molecular weight517.93
Spectral properties
Excitation (nm)420
Emission (nm)505
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 500 is designed for labeling live cells in the same way to calcein, AM. It has a maximum excitation at 405 nm, which perfectly matches the violet laser line equipped in most flow cytometers, and it is well-excited by the excitation sources of fluorescence microscopes. Upon getting into live cells the weakly fluorescent CytoCalcein™ Violet 500 is hydrolyzed into a strongly fluorescent dye that has an excitation/emission maxima of 405/500 nm. This exceptional spectral separation from the typical FACS fluorophores provides additional options for multiplexing experiments. CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 have been developed for flow cytometric applications. CytoCalcein™ dyes exhibit similar biological properties to calcein, AM. They are optimized for the excitation wavelengths of a variety of flow cytometers, providing additional colors for flow cytometric analysis of live cells. CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 are well excited by 405 nm of violet laser and emit fluorescence at 450 nm and 500 nm respectively.


Flow cytometer

Excitation405 nm laser
Emission525/40 nm filter
Instrument specification(s)AmCyan channel

Fluorescence microscope

ExcitationDAPI filter set
EmissionDAPI filter set
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 500 Stock Solution
  1. Prepare a 2 to 5 mM stock solution of CytoCalcein™ Violet 500 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 500 Working Solution
  1. Prepare a CytoCalcein™ Violet 500 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 500 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 500 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 DAPI filter set, a flow cytometer equipped with a violet laser and a 525/40 nm filter (AmCyan channel), or a fluorescence plate reader at Ex/Em = 405/500 nm cutoff 475 nm.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of CytoCalcein™ Violet 500 *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 mM193.076 µL965.381 µL1.931 mL9.654 mL19.308 mL
5 mM38.615 µL193.076 µL386.153 µL1.931 mL3.862 mL
10 mM19.308 µL96.538 µL193.076 µL965.381 µL1.931 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles


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Spectral properties

Excitation (nm)420
Emission (nm)505

Product Family



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