Intracellular Levels of Calcium Assay Using Flow Cytometry

Calcium is an important intracellular messenger in the cell. Intracellular levels of calcium are very low compared with the extracellular environment, and calcium can be stored in intracellular compartments, such as calciosomes, the endoplasmic reticulum, and mitochondria. These intracellular pools can be mobilized by extracellular ligands upon binding of their specific membrane receptors. Extracellular calcium influx is also known to be initiated by membrane depolarization due to an external signal or by the binding of ligand to calcium channels. Certain calcium-binding fluorescent indicators, such as Fluo-3/AM (see Figure 13.1), are membrane permeable and undergo an enhancement of fluorescence on Ca²⁺ binding. Fluo-3 can therefore be used to determine differences in calcium intracellular levels using flow cytometry. Since the Fluo-3 fluorescence is dependent on the intracellular Ca²⁺ concentration, a baseline, reflecting intracellular levels of free calcium of nonactivated cells, is evaluated before any extracellular activator is incorporated into the media. Therefore, the baseline and fluorescence induced by a specific Ca²⁺ activator can be evaluated through flow cytometry. Figure 13.1 Principle of the calcium assay. Lymphocytes are loaded with Fluo-3 AM. which is de-esterified in the cytoplasm. The hydrolyzed Fluo-3 is then nonfluorescent and trapped into the cell. Following an appropriate stimulus, e.g., phytohemaglutinin (PHA), the concentration of intracellular calcium increases. The fluorescence is produced by the binding of Ca²⁺ to Fluo-3 and can be evaluated over time. https://s3-euw1-ap-pe-df-pch-content-public-p.s3.eu-west-1.amazonaws.com/9780429156977/936bbeac-659b-4503-8a0d-08a54ad61dfe/content/fig13_1.tif"/>