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Calcein Orange™ diacetate

Image of Live HeLa cells stained with Calcein Orange™ diacetate (Cat#22009). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).<br> <br> 
Image of Live HeLa cells stained with Calcein Orange™ diacetate (Cat#22009). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).<br> <br> 
Fluorescence image of HeLa cells stained with Calcein Orange™ diacetate in a Costar black wall/clear bottom 96-well plate.
Ordering information
Price ()
Catalog Number22009
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight877.24
SolventDMSO
Spectral properties
Excitation (nm)531
Emission (nm)545
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
877.24
Excitation (nm)
531
Emission (nm)
545
Calcein AM is one of 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 Red™. These new Calcein AM analogs enable the multicolor labeling and functional analysis of live cells in combination with Calcein AM. Non-fluorescent Calcein Orange™ diaceate can easily get into live cells and hydrolyzes to generate strongly fluorescent Calcein Orange™ dye. AAT Bioquest also offers Calcein Orange™ as a reference dye to Calcein Orange™ diacetate.

Platform


Flow cytometer

Excitation488/532 nm laser
Emission575/26 nm filter
Instrument specification(s)PE channel

Fluorescence microscope

ExcitationTRITC filter set
EmissionTRITC filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation520
Emission550
Cutoff530
Recommended plateSolid 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.

Calcein Orange™ Diacetate Stock Solution
Prepare a 2 to 5 mM stock solution of Calcein Orange™ diacetate 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 Orange™ Diacetate Working Solution
Prepare a Calcein Orange™ diacetate working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, Calcein Orange™ diacetate 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 Calcein Orange™ diacetate 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 TRITC filter set, a flow cytometer equipped with a 575/26 nm filter (PE channel), or a fluorescence plate reader at Ex/Em = 520/550 nm cutoff 530 nm. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein Orange™ diacetate 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 mM113.994 µL569.969 µL1.14 mL5.7 mL11.399 mL
5 mM22.799 µL113.994 µL227.988 µL1.14 mL2.28 mL
10 mM11.399 µL56.997 µL113.994 µL569.969 µL1.14 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)531
Emission (nm)545

Citations


View all 18 citations: Citation Explorer
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,
Journal: Cellular signalling (2017): 231--241
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

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
Calcein AM release-based cytotoxic cell assay for fish leucocytes
Authors: Iwanowicz LR, Densmore CL, Ottinger CA.
Journal: Fish Shellfish Immunol (2004): 127
Calcein-AM is a detector of intracellular oxidative activity
Authors: Uggeri J, Gatti R, Belletti S, Sc and roglio R, Corradini R, Rotoli BM, Orl and ini G., undefined
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
Authors: Goto T, Kajiwara H, Yoshinari M, Fukuhara E, Kobayashi S, Tanaka T.
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
Authors: Tokudome Y, Sugibayashi K.
Journal: Biol Pharm Bull (2003): 1508

Application notes


Annexin V