Calcein-AM readily passes through the cell membrane of viable cells because of its enhanced hydrophobicity compared to Calcein. After Calcein-AM permeates into the cytoplasm, it is hydrolyzed by esterases to Calcein, which remains inside the cell (Fig. 1). Among other reagents, including 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 inhibit any 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. The excitation and emission wavelengths of calcein are 490 nm and 515 nm, respectively
Fig. 1 Cell staining mechanism
1.Prepare 1 mM Calcein-AM solution with DMSO and dilute to prepare 1-50 μM Calcein-AM solution with PBS.a)
2.Add Calcein-AM solution with 1/10 of the volume of cell culture medium to the cell culture.b)
3.Incubate the cell at 37ºC for 15-30 min.
4.Wash cells twice with PBS or an appropriate buffer.
5.Observe the cells under a fluorescence microscope with 490 nm excitation and 515 nm emission filters.
a) If the Calcein-AM has difficulty loading into cells, use a detergent such as Pluronic F127.
b) Or you may replace the culture medium with 1/10 concentration of Calcein-AM buffer solution.
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Fig. 2 Cell staining with Calcein-AM
Cell type: HeLa