Calcein UltraGreen™ AM

Images of Live HeLa cells stained with Calcein UltraGreen™, AM (Cat.21905). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).

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Additional ordering information

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

Physical properties

Molecular weight	607.47
Solvent	DMSO

Spectral properties

Excitation (nm)	492
Emission (nm)	514

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

607.47

Excitation (nm)

492

Emission (nm)

514

Calcein UltraGreen™ AM readily passes through the cell membrane of viable cells. Upon transporting into live cells cellular esterases cut off the AM groups, the molecule gets trapped inside cells. Compared with Calcein AM, Calcein UltraGreen™ is more suitable fluorescent probe for staining viable cells because of its lower cytotoxicity and longer retention in cells. UltraGreen™ AM does not significantly affect cellular functions such as proliferation or chemotaxis of lymophocyte.

Platform

Flow cytometer

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Fluorescence microscope

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

Fluorescence microplate reader

Excitation	490
Emission	525
Cutoff	515
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode

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.

Calcein UltraGreen™ AM Stock Solution

Prepare a 2 to 5 mM stock solution of Calcein UltraGreen™ AM 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

Calcein UltraGreen™ AM Working Solution

Prepare a Calcein UltraGreen™ AM working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, Calcein UltraGreen™ AM 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 Calcein UltraGreen™ AM 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 FITC filter set, a flow cytometer equipped with a blue laser and a 530/30 nm filter (FITC channel), or a fluorescence plate reader at Ex/Em = 490/525 nm cutoff 515 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein UltraGreen™ AM 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	164.617 µL	823.086 µL	1.646 mL	8.231 mL	16.462 mL
5 mM	32.923 µL	164.617 µL	329.234 µL	1.646 mL	3.292 mL
10 mM	16.462 µL	82.309 µL	164.617 µL	823.086 µL	1.646 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

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	492
Emission (nm)	514

Product Family

Name	Excitation (nm)	Emission (nm)
Calcein Red™ AM	562	576
Calcein UltraBlue™ AM	359	458
Calcein Blue, AM CAS 168482-84-6	354	441

Images

Figure 1. Fluorescence images of HeLa cells stained with Calcein UltraGreen™ AM (upper row) or Calcein AM (lower row) in a Costar black wall/clear bottom 96-well plate. After washing, growth media were added back, and the cells were monitored using a microscope with FITC filter for up to 24 hours.

Figure 2. Images of Live HeLa cells stained with Calcein UltraGreen™, AM (Cat.21905). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).

Citations

View all 19 citations: Citation Explorer

Dietary oxalate induces urinary nanocrystals in humans
Authors: Kumar, Parveen and Patel, Mikita and Thomas, Vinoy and Knight, John and Holmes, Ross P and Mitchell, Tanecia
Journal: Kidney International Reports (2020): 1040--1051

Establishment and application of a novel fluorescence-based analytical method for the rapid detection of viable bacteria in different samples
Authors: Yin, Qiuyue and Nie, Maiqian and Diwu, Zhenjun and Zhang, Yuting and Wang, Lei and Yin, Dandan and Li, Liancheng
Journal: Analytical Methods (2020): 3933--3943

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

References

View all 84 references: Citation Explorer

Functional evidence that the self-renewal gene NANOG regulates esophageal squamous cancer development
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Journal: Biochemical and Biophysical Research Communications (2017)

Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine
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A vaccination and challenge model using calcein marked fish
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Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging
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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
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Journal: Toxicol In Vitro (2005): 505

Calcein AM release-based cytotoxic cell assay for fish leucocytes
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Journal: Fish Shellfish Immunol (2004): 127

Calcein-AM is a detector of intracellular oxidative activity
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Journal: Histochem Cell Biol (2004): 499

Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines
Authors: Mueller H, Kassack MU, Wiese M.
Journal: J Biomol Screen (2004): 506

In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue
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Journal: Biomaterials (2003): 3885

The effects of calcium chloride and sodium chloride on the electroporation-mediated skin permeation of fluorescein isothiocyanate (FITC)-dextrans in vitro
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