Calcein, AM is a cell staining reagent that can fluorescently label living cells. After it penetrates the cell membrane and enters the cell, it is cleaved by the intracellular esterase to form Calcein, which is retained in the cell and emits strong green fluorescence. Compared with other similar reagents (such as BCECF, AM and CFDA), Calcein, AM has very low cytotoxicity. The excitation and emission wavelengths of Calcein are 490 nm and 515 nm, respectively.
Calcein, AM only stain live cells. PI, which is a nuclear staining dye, cannot pass through the cell membrane of living cells. It passes through the disordered area of the dead cell membrane to reach the nucleus, and is embedded in the cell’s DNA double helix to generate red fluorescence (excitation: 535 nm, emission: 617 nm). PI only stains dead cells. Since both Calcein and PI-DNA can be excited at 490 nm, a fluorescence microscope can be used to observe live and dead cells simultaneously. With 545 nm excitation, only dead cells can be observed. Based on the above characteristics, Calcein, AM and PI are often combined to double stain live and dead cells.
Due to the different optimal staining conditions for different cell lines, we recommend determining the appropriate concentrations of Calcein, AM and PI individually.
Instructions
(1) Prepare 1 mM Calcein, AM solution with DMSO, and dilute it with PBS to make 1-50 μM Calcein, AM solution.
(2) Add 1/10 of the volume of the cell culture medium Calcein, AM solution to the cell culture medium. b)
(3) Incubate the cells at 37°C for 15-30 minutes.
(4) Wash the cells twice with PBS or an appropriate buffer.
(5) Observe the cells with a fluorescence microscope with a filter with excitation wavelength of 490 nm and emission wavelength of 515 nm.
a) If it is difficult for Calcein and AM to enter cells, you can use surfactants such as Pluronic F127.
b) It is also possible to use 1/10 concentration of Calcein, AM solution instead of medium.
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