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).

Images of Live HeLa cells stained with Calcein, AM (Cat.22002).

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 °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: <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.

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Physical properties

Molecular weight	994.86
Solvent	DMSO

Spectral properties

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

Storage, safety and handling

Certificate of Origin	Download PDF
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

Alternative formats

Calcein, AM *UltraPure grade* *CAS 148504-34-1*

Overview SDS Protocol

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.

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 49 citations: Citation Explorer

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Influence of Aerosolization on Endothelial Cells for Efficient Cell Deposition in Biohybrid and Regenerative Applications
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Effervescent Atomizer as Novel Cell Spray Technology to Decrease the Gas-to-Liquid Ratio
Authors: Thiebes, Anja Lena and Klein, Sarah and Zingsheim, Jonas and M{\"o}ller, Georg H and G{\"u}rzing, Stefanie and Reddemann, Manuel A and Behbahani, Mehdi and Jockenhoevel, Stefan and Cornelissen, Christian G
Journal: Pharmaceutics (2022): 2421

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
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Drug/bioactive eluting chitosan composite foams for osteochondral tissue engineering
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Journal: International Journal of Biological Macromolecules (2022)

The effects of acute exercise and inflammation on immune function in early-stage prostate cancer
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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)

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

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
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Journal: Phytomedicine (2021): 153528

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
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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
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Journal: Biol Pharm Bull (2003): 1508