Hello Haiyao Ding

Calcein AM is a non-fluorescent cell-permeable derivate of Calcein that is widely used in cell viability measurement. The carboxylic acid groups on Calcein are modified with AM (acetomethoxy) groups, which endows Calcein AM with high hydrophobicity, facilitating its penetration through cell membrane. Once inside the cell, AM groups are hydrolyzed by intracellular esterases. The fluorescent Calcein molecule is restored, which is trapped in the cell and emits strong green fluorescence.

Since dead cells lack esterase activity, only live cells are labeled and detected. The fluorescence intensity will be proportional to esterase activity. Calcein-AM has been proved to be both specific and sensitive for detection and tracking of apoptosis in living cells. The preservation of membrane integrity is one of the most significant features of apoptosis with respect to necrosis. In the presence of membrane defects, Calcein leaks out of the cell and the signal also vanishes in the presence of residual esterase activity.

On the other hand, Propidium iodide (PI) which is a red-fluorescent nuclear stain is not permeant to live cells or cells which are dead but still have an intact membrane (such as the primary apoptotic cells). In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence.

Calcein generated from esterase in viable cells emits a strong green fluorescence with an excitation and emission maximum at 494nm and 517nm, respectively, while PI once bound to DNA has a maximum emission wavelength at 617nm when excited at 535nm.

There is something that must have gone wrong with your reagent or your process. You may have cells that are either alive or dead, but not both. Cells which are dead but still have an intact membrane (like the primary apoptotic cells), PI is not permeant to these cells.

You may repeat the experiment. Initially, observe the cells in bright field. Then observe the cells in the green fluorescence channel. The live cells will be stained by green, fluorescent Calcein. Follow it by observing the cells in the red fluorescence channel. The dead cells will be stained by the red fluorescent, PI. Then finally you merge the image of green and red channels.

Good Luck!