Calcein+AM

**What is Calcein Acetoxymethyl Ester:**
====Calcein AM is a cell viability indictor used in several applications to pre- and post label viable cells. It is a non-fluorescent compound that can pass through the cell membrane. Once it is absorbed into the cell, it then becomes hydrolyze by the cell's endogenous intracellular esterases into the fluorescent anion, calcein.====



====Calcein AM is commonly used in conjunction with propidium iodide to evaluate its applicability and accurateness. Propidium iodide or ethidium homodimer-1 is generally absorbed when the cells membrane is not completely intact or is compromised. When a red fluorescence appear under a fluorescent microscope it signifies that the cell is dead. The opposite is true for calcein AM. Calcein AM will be absorbed into a cell because of its hydrophobic nature, if the cell is active it will hydrolze this component into calcein and acetoxymethyl ester by its intracellular esterases. The calcein component is hydrophilic so it is not permeable to the membrane and therefore gets trapped inside the cell's cytosol. When it is observed through a fluorescent microscope, it signifies a live cell.====



**How to use Calcein AM:**
====It should be at RT before usage. Cells are usually introduced with this agent by incubation. It is recommended to keep the incubation condition at 37 degrees Celsius and a maximum of 30 minutes to get the optimal results. After incubation the cells can be observed with a fluorescent microscope with the settings at 485-490 nm excitation wavelength and 520-530 nm emission wavelength.====


 * [[image:calceinam:Confocal5_18.jpg width="359" height="305" caption="Confocal Microscope to observe fluorescent dyes"]] ||
 * Confocal Microscope to observe fluorescent dyes ||
 * [[image:calceinam:confocal_mic.jpg width="421" height="322" caption="Fluorescent Microscope System"]] ||
 * Fluorescent Microscope System ||

**How to store Calcein AM:**
====It should be stored at -20 degrees Celsius and protected from light. Avoid exposure to moisture. With these conditions monitored it should remain stable for six months. Without proper storage, aqueous solutions of Calcein AM are susceptible to hydrolyzes. Usage should be within one day. Avoid repeated freeze and thaw cycles.====

-suitable for use in variety of techniques such as: microplate assays, immunocytochemistry, flow cytometry, and in vivo cell tracing

 * Images of Cells Stained with Calcein AM:**
 * [[image:calceinam:green_red_stain.jpg width="587" height="317" caption="Cells labeled with Calcein AM and Propidium iodide"]] ||
 * Cells labeled with Calcein AM and Propidium iodide ||


 * [[image:calceinam:toxcount_fluorescent_micro.jpg width="606" height="327" caption="Cells labeled with Calcein AM and EthD-1"]] ||
 * Cells labeled with Calcein AM and EthD-1 ||


 * [[image:calceinam:bigger_cell.jpg width="624" height="302" caption="A close-up of cells labeled with Calcein AM"]] ||
 * A close-up of cells labeled with Calcein AM ||

References:

Calcein AM. Cell Viability Assay. 2008 R&D Systems, Inc. 

Calcein AM Cell Viability Assay Kit. Biotium, Inc. 

Neri, Simona, E. Mariani, A. Meneghetti, L. Cattini, and A. Facchini. 2001. Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants. Clin Diagn Lab Immunol. 8(6): 1131–1135.

Takenaka, Shoji, H. M. Trivedi, A. Corbin, B. Pitts, and P. S. Stewart. 2008. Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilm. Applied and Environmental Microbiology. 74(6): 1869-1875.