Calcein Deep Red™ acetate

This product is now replaced by #22011. Calcein AM is one the most popular fluorescent probes used for labeling and monitoring cellular functions of live cells. However, the single color of Calcein AM makes it impossible to use this valuable reagent in the multicolor applications. For example, it is impossible to use Calcein AM in combination of GFP-tranfacted cells due to the same color to GFP. To address this color limitation of Calcein AM, we have developed Calcein Orange™, Calcein Red™ and Calcein Deep Red™. These new Calcein AM analogs enable the multicolor labeling and functional analysis of live cells in combination with Calcein AM. Non-fluorescent Calcein Deep Red™ acetate can easily get into live cells and hydrolyzes to generate strongly fluorescent Calcein Deep Red™ (Cat#: 21902) dye. Calcein Deep Red™ dye can be monitored with the common Cy5 filter set.

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 Deep Red™ Acetate Stock Solution

Prepare a 2 to 5 mM stock solution of Calcein Deep Red™ acetate 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 Deep Red™ Acetate Working Solution

Prepare a Calcein Deep Red™ acetate working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, Calcein Deep Red™ acetate 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 Deep Red™ acetate 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 Cy5 filter set, a flow cytometer equipped with a 660/20 nm filter (APC channel), or a fluorescence plate reader at Ex/Em = 620/660 nm cutoff 630 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein Deep Red™ acetate 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	188.551 µL	942.756 µL	1.886 mL	9.428 mL	18.855 mL
5 mM	37.71 µL	188.551 µL	377.102 µL	1.886 mL	3.771 mL
10 mM	18.855 µL	94.276 µL	188.551 µL	942.756 µL	1.886 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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Citations

View all 20 citations: Citation Explorer

Radioiodination of extravesicular surface constituents to study the biocorona, cell trafficking and storage stability of extracellular vesicles
Authors: Yerneni, Saigopalakrishna S and Solomon, Talia and Smith, Jason and Campbell, Phil G
Journal: Biochimica et Biophysica Acta (BBA)-General Subjects (2022): 130069

Bioprinting Exosome-Like Extracellular Vesicle Microenvironments
Authors: Yerneni, Saigopalakrishna S and Whiteside, Theresa L and Weiss, Lee E and Campbell, Phil G
Journal: Bioprinting (2019): e00041

Isoastragaloside I suppresses LPS-induced tight junction disruption and monocyte adhesion on bEnd. 3 cells via an activating Nrf2 antioxidant defense system
Authors: Li, Hong-Li and Jin, Jin-Mei and Yang, Chun and Wang, Ping and Huang, Fei and Wu, Hui and Zhang, Bei-Bei and Shi, Hai-Lian and Wu, Xiao-Jun
Journal: RSC advances (2018): 464--471

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)

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 February 1, 2023

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Catalog Number	22010
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Physical properties

Molecular weight	530.36
Solvent	DMSO

Spectral properties

Excitation (nm)	643
Emission (nm)	663

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