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CytoCalcein™ Violet 500 *Excited at 405 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.

Example protocol

PREPARATION OF STOCK SOLUTIONS

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.

PREPARATION OF WORKING SOLUTION

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.

SAMPLE EXPERIMENTAL PROTOCOL

  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.

Calculators

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.

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Spectrum

Product family

Citations

View all 18 citations: Citation Explorer
Functional imaging of neuronal activity of auditory cortex by using Cal-520 in anesthetized and awake mice
Authors: Li, Jingcheng and Zhang, Jianxiong and Wang, Meng and Pan, Junxia and Chen, Xiaowei and Liao, Xiang
Journal: Biomedical Optics Express (2017): 2599--2610
NINJ2--A novel regulator of endothelial inflammation and activation
Authors: Wang, Jingjing and Fa, Jingjing and Wang, Pengyun and Jia, Xinzhen and Peng, Huixin and Chen, Jing and Wang, Yifan and Wang, Chenhui and Chen, Qiuyun and Tu, Xin and others, undefined
Journal: Cellular Signalling (2017)
Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B
Authors: Bogert, Nicolai V and Werner, Isabella and Kornberger, Angela and Meybohm, Patrick and Moritz, Anton and Keller, Till and Stock, Ulrich A and Beiras-Fern, undefined and ez, Andres
Journal: Scientific reports (2016)
Inhibition of ABC transport proteins by oil sands process affected water
Authors: Alharbi, Hattan A and Saunders, David MV and Al-Mousa, Ahmed and Alcorn, Jane and Pereira, Alberto S and Martin, Jonathan W and Giesy, John P and Wiseman, Steve B
Journal: Aquatic Toxicology (2016): 81--88

References

View all 84 references: Citation Explorer
Functional evidence that the self-renewal gene NANOG regulates esophageal squamous cancer development
Authors: Li, Deng and Xiang, Xiaocong and Yang, Fei and Xiao, Dongqin and Liu, Kang and Chen, Zhu and Zhang, Ruolan and Feng, Gang
Journal: Biochemical and Biophysical Research Communications (2017)
Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine
Authors: Zibek S, Stett A, Koltay P, Hu M, Zengerle R, Nisch W, Stelzle M.
Journal: Biophys J. (2006)
A vaccination and challenge model using calcein marked fish
Authors: Klesius PH, Evans JJ, Shoemaker CA, Pasnik DJ.
Journal: Fish Shellfish Immunol (2006): 20
Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging
Authors: Bratosin D, Mitrofan L, Palii C, Estaquier J, Montreuil J.
Journal: Cytometry A (2005): 78
Cytotoxic effects of 100 reference compounds on Hep G2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I mechanistic assays on ROS, glutathione depletion and calcein uptake
Authors: Schoonen WG, Westerink WM, de Roos JA, Debiton E.
Journal: Toxicol In Vitro (2005): 505
Page updated on December 11, 2024

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Unit size
Catalog Number22013
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Physical properties

Molecular weight

517.93

Solvent

DMSO

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

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

Excitation405
Emission500
Cutoff475
Recommended plateSolid black
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).