AAT Bioquest

Calcein, AM *CAS 148504-34-1*

Simultaneous imaging of live and apoptotic HeLa cells labeled using calcein AM (Cat No. 22002) and Annexin V-iFluor® 647 conjugate (Cat No. 20074).
Simultaneous imaging of live and apoptotic HeLa cells labeled using calcein AM (Cat No. 22002) and Annexin V-iFluor® 647 conjugate (Cat No. 20074).
Simultaneous imaging of live and apoptotic HeLa cells labeled using calcein AM (Cat No. 22002) and Annexin V-iFluor® 647 conjugate (Cat No. 20074).
Images of Live HeLa cells stained with Calcein, AM (Cat.22002). 
Gallery Image 3
Images of Live HeLa cells stained with Calcein, AM (Cat.22002). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
Scheme of experimental temperature modulation. Cells were kept at 37&thinsp;&deg;C initially. (A) HuMEC-1 and PBL were treated for 60&thinsp;min at 37&thinsp;&deg;C. TEM was performed for 120&thinsp;min at 37&thinsp;&deg;C, 30&thinsp;&deg;C and 18&deg;. (B) HuMEC-1 and PBL were treated for 60&thinsp;min at 37&thinsp;&deg;C, 30&thinsp;&deg;C and 18&thinsp;&deg;C. TEM was performed for 120&thinsp;min under the same temperature conditions as HuMEC-1 and PBL were treated before. (C) HuMEC-1 and PBL were treated for 60&thinsp;min at 37&thinsp;&deg;C, 30&thinsp;&deg;C or 18&thinsp;&deg;C with subsequent rewarming to 37&thinsp;&deg;C during TEM for 120&thinsp;min. Source: <strong>Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B</strong> by Bogert et al<em>., Scientific Reports,</em> Feb. 2016.
Ordering information
Catalog Number
Unit Size
Add to cart
Additional ordering information
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Physical properties
Molecular weight994.86
Spectral properties
Extinction coefficient (cm -1 M -1)81000
Excitation (nm)501
Emission (nm)521
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Calcein AM readily passes through the cell membrane of viable cells because of its enhanced hydrophobicity as compared to calcein. The acetomethoxy (AM) derivate of calcein (calcein AM) is widely used for labeling live cells as it can be transported through the cellular membrane into live cells. The AM ester groups mask the part of the molecule that chelates calcium. Upon transporting into live cells cellular esterases cut off the AM groups, the molecule binds to calcium within cell (resulting in acquiring strong green fluorescence), and gets trapped inside. As dead cells lack esterases, only live cells are marked. This feature makes it very useful for testing of cell viability and for short-term marking of cells. Compared with other live cell-labeling reagents (such as BCECF-AM and carboxy-fluorescein diacetate), calcein-AM is the most suitable fluorescent probe for staining viable cells because of its low cytotoxicity. Calcein does not significantly affect cellular functions such as proliferation or chemotaxis of lymophocyte. In addition, viability assays using calcein are reliable and correlate well with the standard 51Cr-release assay.


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

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Example protocol


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

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


Calcein AM working solution
  1. Prepare a Calcein 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 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.


  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 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.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein, AM *CAS 148504-34-1* 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 mM100.517 µL502.583 µL1.005 mL5.026 mL10.052 mL
5 mM20.103 µL100.517 µL201.033 µL1.005 mL2.01 mL
10 mM10.052 µL50.258 µL100.517 µL502.583 µL1.005 mL

Molarity calculator

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

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


Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm -1 M -1)81000
Excitation (nm)501
Emission (nm)521



View all 54 citations: Citation Explorer
Elucidating the distinctive regulatory effects and mechanisms of active compounds in Salvia miltiorrhiza Bunge via network pharmacology: Unveiling their roles in the modulation of platelet activation and thrombus formation
Authors: Zhang, Ying and Xin, Guang and Zhou, Qilong and Yu, Xiuxian and Feng, Lijuan and Wen, Ao and Zhang, Kun and Wen, Tingyu and Zhou, Xiaoli and Wu, Qiuling and others,
Journal: Toxicology and Applied Pharmacology (2024): 116871
Reinforced Immunogenic Endoplasmic Reticulum Stress and Oxidative Stress via an Orchestrated Nanophotoinducer to Boost Cancer Photoimmunotherapy
Authors: Yang, Zhenzhen and Teng, Yulu and Lin, Meng and Peng, Yiwei and Du, Yitian and Sun, Qi and Gao, Datong and Yuan, Quan and Zhou, Yu and Yang, Yiliang and others,
Journal: ACS nano (2024)
Type I collagen and fibromodulin enhance the tenogenic phenotype of hASCs and their potential for tendon regeneration
Authors: Tu, Tian and Shi, Yuan and Zhou, Boya and Wang, Xiaoyu and Zhang, Wenjie and Zhou, Guangdong and Mo, Xiumei and Wang, Wenbo and Wu, Jinglei and Liu, Wei
Journal: npj Regenerative Medicine (2023): 67
Hyperglycemia-regulated tRNA-derived fragment tRF-3001a propels neurovascular dysfunction in diabetic mice
Authors: Zhu, Jun-Ya and Yao, Wen and Ni, Xi-Sen and Yao, Mu-Di and Bai, Wen and Yang, Tian-Jing and Zhang, Zi-Ran and Li, Xiu-Miao and Jiang, Qin and Yan, Biao
Journal: Cell Reports Medicine (2023)
3D Spheroid Cultivation Alters the Extent and Progression of Osteogenic Differentiation of Mesenchymal Stem/Stromal Cells Compared to 2D Cultivation
Authors: Wolff, Anne and Frank, Marcus and Staehlke, Susanne and Springer, Armin and Hahn, Olga and Meyer, Juliane and Peters, Kirsten
Journal: Biomedicines (2023): 1049
Influence of Aerosolization on Endothelial Cells for Efficient Cell Deposition in Biohybrid and Regenerative Applications
Authors: Cheremkhina, Maria and Klein, Sarah and Babendreyer, Aaron and Ludwig, Andreas and Schmitz-Rode, Thomas and Jockenhoevel, Stefan and Cornelissen, Christian G and Thiebes, Anja Lena
Journal: Micromachines (2023): 575
Drug/bioactive eluting chitosan composite foams for osteochondral tissue engineering
Authors: Samie, Muhammad and Khan, Ather Farooq and Rahman, Saeed Ur and Iqbal, Haffsah and Yameen, Muhammad Arfat and Chaudhry, Aqif Anwar and Galeb, Hanaa A and Halcovitch, Nathan R and Hardy, John G
Journal: International Journal of Biological Macromolecules (2022)
Regulatory effect of long-stranded non-coding RNA-CRNDE on neurodegeneration during retinal ischemia-reperfusion
Authors: Sun, Ting-Ting and Li, Xiu-Miao and Zhu, Jun-Ya and Yao, Wen and Yang, Tian-Jing and Meng, Xiang-Rui and Yao, Jin and Jiang, Qin
Journal: Heliyon (2022): e10994
Sensing of Physical Factors by Cells: Electric Field, Mechanical Forces, Physical Plasma and Light—Importance for Tissue Regeneration
Authors: Bielfeldt, Meike and Rebl, Henrike and Peters, Kirsten and Sridharan, Kaarthik and Staehlke, Susanne and Nebe, J Barbara
Journal: Biomedical Materials \& Devices (2022): 1--16


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