Calcein UltraBlue™ AM

Calcein UltraBlue™ AM is a cell-permeable version of Calcein UltraBlue™. Upon getting into live cells the weakly fluorescent Calcein UltraBlue™ AM is hydrolyzed into Calcein UltraBlue™ that has the excitation/emission maxima similar to those of Calcein Blue, DAPI, Hoechst and AMCA. This exceptional spectral separation from the typical green and red fluorophores (such as FITC, TMR and Texas Red) provides additional options for multiplexing experiments. Calcein UltraBlue™ has similar spectral properties to those of Calcein Blue. However, compared with Calcein Blue, Calcein UltraBlue™ has higher photostability and stronger fluorescence intensity at physiological pH, making it a more robust fluorescent probe than Calcein Blue.

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 UltraBlue™ AM Stock Solution

Prepare a 2 to 5 mM stock solution of Calcein UltraBlue™ 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 UltraBlue™ AM Working Solution

Prepare a Calcein UltraBlue™ 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 UltraBlue™ 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 UltraBlue™ 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 DAPI filter set, a flow cytometer equipped with a UV/violet laser and a 450/40 nm filter (Pacific Blue channel), or a fluorescence plate reader at Ex/Em = 360/450 nm cutoff 420 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein UltraBlue™ 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	124.583 µL	622.913 µL	1.246 mL	6.229 mL	12.458 mL
5 mM	24.917 µL	124.583 µL	249.165 µL	1.246 mL	2.492 mL
10 mM	12.458 µL	62.291 µL	124.583 µL	622.913 µL	1.246 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

Product family

Name	Excitation (nm)	Emission (nm)
Calcein Red™ AM	562	576
Calcein Blue, AM CAS 168482-84-6	354	441
Calcein UltraGreen™ AM	492	514

Citations

View all 19 citations: Citation Explorer

The zebrafish heart harbors a thermogenic beige fat depot analog of human epicardial adipose tissue
Authors: Morocho-Jaramillo, Paul-Andres and Kotlar-Goldaper, Ilan and Zakarauskas-Seth, Bhakti I and Purf{\"u}rst, Bettina and Filosa, Alessandro and Sawamiphak, Suphansa
Journal: Cell Reports (2024)

Double emulsion picoreactors for high-throughput single-cell encapsulation and phenotyping via FACS
Authors: Brower, Kara K and Khariton, Margarita and Suzuki, Peter H and Still, Chris and Kim, Gaeun and Calhoun, Suzanne GK and Qi, Lei S and Wang, Bo and Fordyce, Polly M
Journal: Analytical chemistry (2020): 13262--13270

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)

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 3, 2024

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Catalog Number	21908
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Physical properties

Molecular weight	802.68
Solvent	DMSO

Spectral properties

Excitation (nm)	359
Emission (nm)	458

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