Intracellular increase of calcium concentration is a very common early response to external stimulation of many different cell types. Calcium enters the cytoplasm from intracellular calcium stores in the endoplasmic reticulum (ER) as well as from the extracellular space through membrane bound calcium ion channels.
Calcium dyes bind calcium ions and change their fluorescent properties/intensity. This change is detected by the cytometer over time and allows the generation of kinetic calcium curves. There are mainly two different kind of calcium dyes. Non-ratiometric calcium dyes, like Fluo-3, increase their fluorescent intensity when bound to calcium. The increase of fluorescence can be correlated to the amount of calcium that entered the cell over time. Ratiometric dyes, like Indo-1 change their fluorescent properties when bound to calcium. “Free” Indo-1 emits at ~530 nm and binding to calcium shifts the emission to ~390 nm. The calculation of relative ratios of bound versus unbound Indo-1 allows for a much more sensitive detection of changes in calcium concentration.
The main advantages of using flow cytometry for calcium analysis are the increased signal sensitivity and much higher throughput compared to fluorescence microscopy. Furthermore, the multiparameter analysis allows to run internal negative controls or compare the calcium flux ability of several subpopulations within one cell sample in one experiment.
The combination of optogenetics, calcium and flow has been successfully achieved by our pxONE user family members Sascha Yousefi and Birthe Stüven.