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Fluorescent Dye AM Esters

Introduction



The principle of Fluo-3 AM is loaded into live cell for intracellular Ca2+ detection.
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


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


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


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

Table 1. UV-Excitable Calcium Indicators

Ca2+ Indicator
Product Numbers
Molecular Weight
Ex
Em
Calcein blue, AM22007465.41360 nm445 nm
CytoCalcein Violet 45022012400408 nm460 nm
Fura-2, AM21020, 21021, 21022, 210231001.86340/380 nm510 nm
Indo-1, AM21030, 21032, 21033, 210361009.91355 nm400/475 nm
Quin-2, AM21050829.76346 nm475 nm

Table 2. Visible Light-Excitable Calcium Indicators

Ca2+ Indicator
Product Numbers
Molecular Weight
Ex
Em
Calcein, AM22002, 22003, 22004994.86490 nm515 nm
Fluo-3, AM21010, 21011, 21012, 21013, 210141129.85506 nm526 nm
Fluo-8®, AM21080, 21081, 21082, 210831000490 nm514 nm
Fluo-8H™, AM21090, 210911100490 nm514 nm
Fluo-8L™, AM21096, 210971100490 nm514 nm
Rhod-4™, AM21120, 21121, 21122, 211231000530 nm555 nm
Rhod-2, AM21060, 21062, 21063, 21064 1123.96549 nm578 nm
Rhod-5N, AM210701154.92551 nm577 nm

Table 3. Non-Fluorescent Calcium Detection AM esters

Ca2+ Indicator
Product Numbers
Molecular Weight
BAPTA, AM21001, 21002764.68
EGTA AM21005, 21006668.6

Table 4. Other Ion Fluorescent Indicators

Ca2+ Indicator
Product Numbers
Molecular Weight
Ex
Em
BCECF, AM21202, 21203, 21204764.68505 nm520 nm

 

References


  1. Zibek S, Stett A, Koltay P, Hu M, Zengerle R, Nisch W, Stelzle M. (2006) Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine. Biophys J.
  2. Klesius PH, Evans JJ, Shoemaker CA, Pasnik DJ. (2006) A vaccination and challenge model using calcein marked fish. Fish Shellfish Immunol, 20, 20.
  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.


Original created on July 23, 2012, last updated on July 23, 2012
Tagged under: Cell Viability, Proliferation & Function