Calcein, AM *CAS 148504-34-1*
| Overview |  SDSProtocol |
Upgrade: Calcein UltraGreen™ AM
CAS 148504-34-1 | Molecular weight 994.86 | Extinction coefficient (cm -1 M -1) 81000 | Excitation (nm) 501 | Emission (nm) 521 |
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.
Platform
Flow cytometer
| Excitation | 488 nm laser |
| Emission | 530/30 nm filter |
| Instrument specification(s) | FITC channel |
Fluorescence microscope
| Excitation | FITC filter set |
| Emission | FITC filter set |
| Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
| Excitation | 490 |
| Emission | 525 |
| Cutoff | 515 |
| Recommended plate | Black wall/clear bottom |
| Instrument specification(s) | Bottom read mode |
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.
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.
Calcein AM stock solution
Prepare a 2 to 5 mM stock solution of Calcein 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 AM working solution
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 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 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 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.
Calculators
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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 100.517 µL | 502.583 µL | 1.005 mL | 5.026 mL | 10.052 mL |
| 5 mM | 20.103 µL | 100.517 µL | 201.033 µL | 1.005 mL | 2.01 mL |
| 10 mM | 10.052 µL | 50.258 µL | 100.517 µL | 502.583 µL | 1.005 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
Spectral properties
| Extinction coefficient (cm -1 M -1) | 81000 |
| Excitation (nm) | 501 |
| Emission (nm) | 521 |
Images
Figure 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).
Figure 2. Images of Live HeLa cells stained with Calcein, AM (Cat.22002).
Figure 3. Chemical structure for Calcein, AM *CAS 148504-34-1*
Figure 4. Images of Live HeLa cells stained with Calcein, AM (Cat.22002). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
Figure 5. Scheme of experimental temperature modulation. Cells were kept at 37 °C initially. (A) HuMEC-1 and PBL were treated for 60 min at 37 °C. TEM was performed for 120 min at 37 °C, 30 °C and 18°. (B) HuMEC-1 and PBL were treated for 60 min at 37 °C, 30 °C and 18 °C. TEM was performed for 120 min under the same temperature conditions as HuMEC-1 and PBL were treated before. (C) HuMEC-1 and PBL were treated for 60 min at 37 °C, 30 °C or 18 °C with subsequent rewarming to 37 °C during TEM for 120 min. Source: Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B by Bogert et al., Scientific Reports, Feb. 2016.
Citations
View all 42 citations: Citation Explorer
pH-responsive biopolymer-based supramolecular architectures as biodegradable carriers for 1 DOX delivery 2
Authors: Nia, Marzieh Heidari and Ashkar, Said and Munguia-Lopez, Jose G and Kinsella, Joseph M and van de Ven, Theo GM
Journal: (2021)
Authors: Nia, Marzieh Heidari and Ashkar, Said and Munguia-Lopez, Jose G and Kinsella, Joseph M and van de Ven, Theo GM
Journal: (2021)
Highly Concentrated Nitrogen-Doped Carbon Nanotubes in Alginate--Gelatin 3D Hydrogels Enable in Vitro Breast Cancer Spheroid Formation
Authors: Munguia-Lopez, Jose G and Jiang, Tao and Ferlatte, Audrey and Fajardo-Diaz, Juan L and Munoz-Sandoval, Emilio and Tran, Simon D and Kinsella, Joseph M
Journal: Advanced NanoBiomed Research (2021): 2100104
Authors: Munguia-Lopez, Jose G and Jiang, Tao and Ferlatte, Audrey and Fajardo-Diaz, Juan L and Munoz-Sandoval, Emilio and Tran, Simon D and Kinsella, Joseph M
Journal: Advanced NanoBiomed Research (2021): 2100104
A novel flavonoid from Fissistigma cupreonitens, 5-hydroxy-7, 8-dimethoxyflavanone, competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance
Authors: Teng, Yu-Ning and Lin, Kun-I and Lin, Yu-Chao and Thang, Tran-Dinh and Lan, Yu-Hsuan and Hung, Chin-Chuan
Journal: Phytomedicine (2021): 153528
Authors: Teng, Yu-Ning and Lin, Kun-I and Lin, Yu-Chao and Thang, Tran-Dinh and Lan, Yu-Hsuan and Hung, Chin-Chuan
Journal: Phytomedicine (2021): 153528
The COL-4A1 polypeptide destroy endothelial cells through the TGF-$\beta$/PI3K/AKT pathway
Authors: Li, Ting and Ling, Zhonghui and Xie, Kaipeng and Wang, Yixiao and Miao, Zhijing and Ji, Xiaohong and Li, Jingyun and Hou, Wenwen and Tang, Qiuqin and Yuan, Xiaojie and others,
Journal: Scientific Reports (2021): 1--10
Authors: Li, Ting and Ling, Zhonghui and Xie, Kaipeng and Wang, Yixiao and Miao, Zhijing and Ji, Xiaohong and Li, Jingyun and Hou, Wenwen and Tang, Qiuqin and Yuan, Xiaojie and others,
Journal: Scientific Reports (2021): 1--10
On the relevance of ligament dynamics for cell survival in air-assisted bronchoscopic spray application
Authors: G{\"u}rzing, Stefanie and Klein, Sarah and M{\"o}ller, Georg and Thiebes, Anja Lena and Cornelissen, Christian Gabriel and Reddemann, Manuel Armin
Journal: Atomization and Sprays (2021)
Authors: G{\"u}rzing, Stefanie and Klein, Sarah and M{\"o}ller, Georg and Thiebes, Anja Lena and Cornelissen, Christian Gabriel and Reddemann, Manuel Armin
Journal: Atomization and Sprays (2021)
A novel peptide ameliorates TNF$\alpha$-and LPS-induced endothelia dysfunction in preeclampsia
Authors: Ji, Xiaohong and Li, Chanjuan and Lv, Yan and Miao, Zhijing and Wu, Lan and Long, Wei and Wang, Xing and Ding, Hongjuan
Journal: American Journal of Hypertension (2021)
Authors: Ji, Xiaohong and Li, Chanjuan and Lv, Yan and Miao, Zhijing and Wu, Lan and Long, Wei and Wang, Xing and Ding, Hongjuan
Journal: American Journal of Hypertension (2021)
Caffeic Acid Attenuates Multi-Drug Resistance in Cancer Cells by Inhibiting Efflux Function of Human P-glycoprotein
Authors: Teng, Yu-Ning and Wang, Charles CN and Liao, Wei-Chieh and Lan, Yu-Hsuan and Hung, Chin-Chuan
Journal: Molecules (2020): 247
Authors: Teng, Yu-Ning and Wang, Charles CN and Liao, Wei-Chieh and Lan, Yu-Hsuan and Hung, Chin-Chuan
Journal: Molecules (2020): 247
Restoration of MARCK enhances chemosensitivity in cancer
Authors: Wenzel, Tim and B{\"u}ch, Thomas and Urban, Nicole and Weirauch, Ulrike and Schierle, Katrin and Aigner, Achim and Schaefer, Michael and Kalwa, Hermann
Journal: Journal of Cancer Research and Clinical Oncology (2020): 1--16
Authors: Wenzel, Tim and B{\"u}ch, Thomas and Urban, Nicole and Weirauch, Ulrike and Schierle, Katrin and Aigner, Achim and Schaefer, Michael and Kalwa, Hermann
Journal: Journal of Cancer Research and Clinical Oncology (2020): 1--16
Cys-peptide mediates the protective role in preeclampsia-like rat and cell models
Authors: Ji, Xiaohong and Wang, Xing and Ling, Zhonghui and Lv, Yan and Yu, Wei and Jia, Ruizhe and Ding, Hongjuan
Journal: Life Sciences (2020): 117625
Authors: Ji, Xiaohong and Wang, Xing and Ling, Zhonghui and Lv, Yan and Yu, Wei and Jia, Ruizhe and Ding, Hongjuan
Journal: Life Sciences (2020): 117625
Bi-layered α-tocopherol acetate loaded membranes for potential wound healing and skin regeneration
Authors: Zahid, Saba and Khalid, Hamad and Ikram, Fakhera and Iqbal, Haffsah and Samie, Muhammad and Shahzadi, Lubna and Shah, Asma Tufail and Yar, Muhammad and Chaudhry, Aqif Anwar and Awan, Sana Javaid and others, undefined
Journal: Materials Science and Engineering: C (2019)
Authors: Zahid, Saba and Khalid, Hamad and Ikram, Fakhera and Iqbal, Haffsah and Samie, Muhammad and Shahzadi, Lubna and Shah, Asma Tufail and Yar, Muhammad and Chaudhry, Aqif Anwar and Awan, Sana Javaid and others, undefined
Journal: Materials Science and Engineering: C (2019)
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)
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)
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
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
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
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
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
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
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
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
Authors: Tokudome Y, Sugibayashi K.
Journal: Biol Pharm Bull (2003): 1508
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