Introduction

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. 

Product Size

IC-1556-50/50ug

IC-1556-250/250ug

Safety Information

Please wear gloves, lab coat and goggles while operating. Prevent contact product directly. In case of contacting, wash with large amount of water.

Storage

Western Blot HRP Substrate should be stored at -20 ¡ãC and shielded from light. The product will be stable for 12 months under -20¡æ

Platform

Example protocol

A.PREPARATION OF STOCK SOLUTIONS

 Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 ¡ãC after preparation. Avoid repeated freeze-thaw cycles.

Prepare a 2 to 5 mM stock solution of Calcein AM in high-quality, anhydrous DMSO.

B. PREPARATION OF WORKING SOLUTION

Prepare a Calcein AM working solution of 1 to 10 ¦ÌM 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 ¦ÌM 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¨C2.5 mM) or sulfinpyrazone (0.1¨C0.25 mM) may be added to the working solution to reduce leakage of the de-esterified indicators.

C. SAMPLE EXPERIMENTAL PROTOCOL

1.Prepare cells for imaging.
2.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.
3.Add Calcein AM working solution to the culture.
4.Incubate cells at 37 ¡ãC for 30 to 60 minutes.
5.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.
6.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. 

InCellGene LLC

A Brand Company Of BioCytoSci((BCS) INC.

Website: www.incellgene.com