CytoCalcein™ Violet 500 Excited at 405 nm

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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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
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Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	517.93
Solvent	DMSO

Spectral properties

Excitation (nm)	420
Emission (nm)	505

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Molecular weight

517.93

Excitation (nm)

420

Emission (nm)

505

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.

Platform

Flow cytometer

Excitation	405 nm laser
Emission	525/40 nm filter
Instrument specification(s)	AmCyan channel

Fluorescence microscope

Excitation	DAPI filter set
Emission	DAPI filter set
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	405
Emission	500
Cutoff	475
Recommended plate	Solid black

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

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

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

Prepare cells for imaging.
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.
Add CytoCalcein™ Violet 500 working solution to the culture.
Incubate cells at 37 °C for 30 to 60 minutes.
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.
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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	193.076 µL	965.381 µL	1.931 mL	9.654 mL	19.308 mL
5 mM	38.615 µL	193.076 µL	386.153 µL	1.931 mL	3.862 mL
10 mM	19.308 µL	96.538 µL	193.076 µL	965.381 µL	1.931 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

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Spectral properties

Excitation (nm)	420
Emission (nm)	505

Product Family

Name	Excitation (nm)	Emission (nm)
CytoCalcein™ Violet 450 Excited at 405 nm	406	445
ThiolTrace™ Violet 500	415	499
CytoTell™ Violet 500	415	499

Images

Figure 1. Fluorescence image of HeLa cells stained with CytoCalcein™ Violet 500 *Excited at 405 nm* in a Costar black wall/clear bottom 96-well plate.

Figure 2. Images of Live HeLa cells stained with CytoCalcein Violet 500 (Cat.22013). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).

Citations

View all 17 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

Rapid generation of collagen-based microtissues to study cell--matrix interactions
Authors: Brett, Marie-Elena and Crampton, Alex and ra L , undefined and Wood, David K
Journal: Technology (2016): 1--8

Toxicokinetics and toxicodynamics of chlorpyrifos is altered in embryos of Japanese medaka exposed to oil sands process-affected water: evidence for inhibition of P-glycoprotein
Authors: Alharbi, Hattan A and Alcorn, Jane and Al-Mousa, Ahmed and Giesy, John P and Wiseman, Steve B
Journal: Journal of Applied Toxicology (2016)

Flexible Endoscopic Spray Application of Respiratory Epithelial Cells as Platform Technology to Apply Cells in Tubular Organs
Authors: Thiebes, Anja Lena and Reddemann, Manuel Armin and Palmer, Johannes and Kneer, Reinhold and Jockenhoevel, Stefan and Cornelissen, Christian Gabriel
Journal: Tissue Engineering Part C: Methods (2016): 322--331

Erythropoietin Stimulates Endothelial Progenitor Cells to Induce Endothelialization in an Aneurysm Neck After Coil Embolization by Modulating Vascular Endothelial Growth Factor
Authors: Liu, Peixi and Zhou, Yingjie and An, Qingzhu and Song, Yaying and Chen, Xi and Yang, Guo-Yuan and Zhu, Wei
Journal: MEDICINE (2016): 1--8

Spraying respiratory epithelial cells to coat tissue-engineered constructs
Authors: Thiebes, Anja Lena and Albers, Stefanie and Klopsch, Christian and Jockenhoevel, Stefan and Cornelissen, Christian G
Journal: BioResearch open access (2015): 278--287

Visualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions
Authors: Le Bihan, Olivier and Decossas, Marion and Gontier, Etienne and Gerbod-Giannone, Marie-Christine and Lambert, Olivier
Journal: Journal of structural biology (2015): 470--477

References

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