Calcein Blue is a blue-fluorescent, short-term dye.

Calcein blue AM is only weakly fluorescent (excitation/emission maxima 322/435 nm). Upon cleavage of the AM esters by intracellular esterases, this tracer becomes relatively polar and is retained by cells for several hours. In addition, its fluorescence intensity increases, and its fluorescence spectra shift to longer wavelengths, with excitation/emission maxima of 360/449 nm [1]. When dopamine is added to a solution of the calcein blue–Fe2 complex, dopamine–Fe2 complex is formed as the result of a ligand exchange reaction between calcein blue and dopamine which permits the fluorescence from calcein blue to be recovered. The fluorescence intensity at the wavelength of 440 nm (at the excitation wavelength of 340 nm) is found to be proportional to the concentration of the dopamine added to the calcein blue–Fe2 complex solution, which permits dopamine to be quantitatively determined [2].

Instructions
I. Solution preparation
1. Preparation of mother solution: Dissolve Calcein Blue AM in high-quality anhydrous DMSO to prepare a 1-5 mM stock solution. It is recommended to prepare a high-concentration stock solution to reduce the DMSO content in the solution (ideally ≤0.1%).
Notes: The prepared storage, well sealed, and frozen under dry conditions, the stock solution can be stored stably for at least 6 months under appropriate conditions.
2. Preparation of working solution: Dilute the stock solution to the required working concentration using an appropriate buffer (such as PBS or HBSS-HEPES), usually 2-5 μM.
Notes: Since the optimal staining conditions for different cell lines are different, it is recommended to perform a gradient experiment to determine the optimal working concentration.
II. Operation steps
1. Cell staining:
Adherent cells: Digest the cells with trypsin-EDTA and collect them by centrifugation (1000 rpm, 3 minutes).
Suspended cells: Collect the cells directly by centrifugation.
1) Wash the cells 2-3 times with PBS or other buffer to remove residual enzyme activity.
2) Resuspend the cells with the staining working solution and incubate at 37°C for 20-60 minutes. The incubation time can be adjusted according to the specific experimental requirements.
2. Washing: After incubation, remove the staining working solution and wash the cells 2-3 times with PBS or appropriate buffer to remove unbound dye.
3. Detection: Use a fluorescence microscope or flow cytometer for detection. The excitation wavelength of Calcein Blue is 360 nm and the emission wavelength is 445 nm.
Notes:
1) Wear gloves during operation to avoid contact between skin or mucous membranes and reagents.
2) Avoid light during incubation and storage to prevent fluorescence quenching.
3) After staining, fluorescence detection analysis should be performed immediately.