Fluorescent Dye AM Esters

Introduction

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Fluorescent dye AM esters are hydrophobic compounds that easily permeates intact live cells, and are widely used for loading a variety of polar fluorescent probes into live cells non-invasively. The nonpolar AM esters readily cross live cell membranes, and rapidly hydrolyzed by cellular esterases inside live cells. The hydrolysis of the esterified groups is essential for analyzing a variety of cellular functions. In some cases (e.g., calcein AM), the AM ester is colorless and non-fluorescent until hydrolyzed.

Storage Conditions

Store at –20⁰C, protected from light. Expiration date is 6 months from the date of receipt.

 

Prepare the AM Ester Stock Solutions

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A) Prepare a 1 to 10 mM stock solution of AM esters just before use in high quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions should be stored well sealed, kept desiccated at - 20°C and protected from light. Under these conditions, AM esters should be stable for several months.

Note: AM esters are susceptible to hydrolysis, particularly in solution. So keep the AM ester stock solutions as concentrated as possible for optimal results. Minimum water content of DMSO (ideally ≤ 0.1%) should be added into the loading solution.

B) The nonionic detergent Pluronic^® F-127 is sometimes used to increase the aqueous solubility of AM esters.

Note: An equal volume of 20% Pluronic^® F-127 solution can be added to DMSO stock solutions before diluting into the loading buffer. The final Pluronic^® F-127 concentration is about 0.02%. A variety of Pluronic^® F-127 solutions can be purchased from AAT Bioquest. The long-term storage of AM esters in the presence of Pluronic^® F-127 is not recommended.

Loading Cells with AM Esters

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Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.

A) On the day of the experiment, either dissolve AM esters in DMSO or thaw an aliquot of the indicator stock solutions to room temperature. Prepare a working solution of 1 to 10 µM (higher concentrations of weakly fluorescent indicators may be required) in the buffer of your choice (such as Hanks and Hepes buffer). For most of cell lines, AM esters (4-5 µM) are recommended. The exact concentration of indicators required for cell loading must be determined empirically. To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal dye concentration that can generate sufficient signal strength.

B) If the cells containing the organic anion-transports, probenecid (1-2.5 mM) or sulfinpyrazone (0.1- 0.25 mM) may be added to the cell medium to reduce leakage of the de-esterified indicators. Incubate cells with the AM esters for 20 minutes to one hour (Longer incubation time is required for weakly fluorescent indicators) at room temperature or 37°C.

Note: Decreasing the loading temperature might reduce the compartmentalization of the indicators.

C) Wash cells in dye-free buffers (containing an anion transporter inhibitor, if applicable) to remove excess probes.

D) Measure fluorescence intensity at desired Ex/Em wavelengths.

AM Ester Products

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The following tables summarized current AM ester products from AAT Bioquest.

References

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3. Bratosin D, Mitrofan L, Palii C, Estaquier J, Montreuil J. (2005) Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging. Cytometry A, 66, 78.
4. Schoonen WG, Westerink WM, de Roos JA, Debiton E. (2005) 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. Toxicol In Vitro, 19, 505.
5. Uggeri J, Gatti R, Belletti S, Scandroglio R, Corradini R, Rotoli BM, Orlandini G. (2004) CalceinAM is a detector of intracellular oxidative activity. Histochem Cell Biol, 122, 499.
6. Mueller H, Kassack MU, Wiese M. (2004) Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines. J Biomol Screen, 9, 506.
7. Iwanowicz LR, Densmore CL, Ottinger CA. (2004) Calcein AM release-based cytotoxic cell assay for fish leucocytes. Fish Shellfish Immunol, 16, 127.
8. Tokudome Y, Sugibayashi K. (2003) The synergic effects of various electrolytes and electroporation on the in vitro skin permeation of calcein. J Control Release, 92, 93.