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Calcein UltraGreen™ AM

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.

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
  1. Prepare a 2 to 5 mM stock solution of Calcein UltraGreen™ AM in high-quality, anhydrous DMSO.

    Note: When reconstituted in DMSO, Calcein UltraGreen™ AM is a clear, colorless solution.

PREPARATION OF WORKING SOLUTION

Calcein UltraGreen™ AM Working Solution
  1. 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: 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.

    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. A variety of ReadiUse™ Probenecid products, Including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest.

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 UltraGreen™ AM 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 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.

Spectrum

Product family

NameExcitation (nm)Emission (nm)
Calcein Red™ AM562576
Calcein UltraBlue™ AM359458
Calcein Blue, AM *CAS 168482-84-6*354441

Citations

View all 20 citations: Citation Explorer
Investigation into the role of H2-Ab1 in vascular remodeling in pulmonary arterial hypertension via Bioinformatics
Authors: Wang, Guowen and Wang, Zhuoyan
Journal: BMC Pulmonary Medicine (2024): 1--12
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
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
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 October 11, 2024

Ordering information

Price
Unit size
Catalog Number21905
Quantity
Add to cart

Additional ordering information

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Physical properties

Molecular weight

607.47

Solvent

DMSO

Spectral properties

Excitation (nm)

492

Emission (nm)

514

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Flow cytometer

Excitation488 nm laser
Emission530, 30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation490
Emission525
Cutoff515
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode
Fixability of live HeLa cells stained with Calcein AM and Calcein UltraGreen AM. HeLa cells were stained with Calcein AM (Catalog #22004) and Calcein UltraGreen AM (Catalog #21905) and then fixed in 4% formaldehyde. Image acquisition was performed on a fluorescence microscope equipped with a FITC filter set. Notably, staining achieved with Calcein UltraGreen AM demonstrated prolonged stability post-fixation, indicating its effectiveness for long-term cellular imaging applications.
Fixability of live HeLa cells stained with Calcein AM and Calcein UltraGreen AM. HeLa cells were stained with Calcein AM (Catalog #22004) and Calcein UltraGreen AM (Catalog #21905) and then fixed in 4% formaldehyde. Image acquisition was performed on a fluorescence microscope equipped with a FITC filter set. Notably, staining achieved with Calcein UltraGreen AM demonstrated prolonged stability post-fixation, indicating its effectiveness for long-term cellular imaging applications.
Fixability of live HeLa cells stained with Calcein AM and Calcein UltraGreen AM. HeLa cells were stained with Calcein AM (Catalog #22004) and Calcein UltraGreen AM (Catalog #21905) and then fixed in 4% formaldehyde. Image acquisition was performed on a fluorescence microscope equipped with a FITC filter set. Notably, staining achieved with Calcein UltraGreen AM demonstrated prolonged stability post-fixation, indicating its effectiveness for long-term cellular imaging applications.
Fluorescence images of HeLa cells stained with Calcein UltraGreen&trade; 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.
Images of Live HeLa cells stained with Calcein UltraGreen&trade;, AM (Cat.21905). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).