Calcium Imaging in H4IIE Liver Cells and Primary Rodent Hepatocytes: A Cost-Effective Protocol for Use With Fura-2 AM...

Calcium signaling is a universal, versatile process in which ionized or free calcium (Ca²⁺) acts as a second messenger to regulate various cellular activities, including hormone secretion, contraction, proliferation, gene expression, and apoptosis. Changes in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) in hepatocytes play a central role in mediating the actions of insulin, glucagon, catecholamines, and other hormones on carbohydrate, lipid, and protein metabolism in the liver. Ratiometric chemical Ca²⁺ indicators are fluorescent dyes that change their emission or excitation spectrum upon binding to calcium, allowing for precise, quantitative measurements of changes in the intracellular Ca²⁺ concentration. They enable calibration by calculating the ratio of two fluorescence intensities, correcting for artifacts such as uneven dye loading, photobleaching, and cell volume variations. Fura-2 acetoxymethyl ester (AM) (hereinafter referred to as Fura-2), a ratiometric and sensitive indicator dye, is a popular fluorescent Ca²⁺ reporter for measuring intracellular calcium. Here, we describe a comprehensive and detailed protocol for Ca²⁺ imaging of the H4IIE cell line and primary rodent hepatocytes in vitro via the chemical reporter Fura-2, which can also be employed on a wide variety of cell types. Unlike previously published protocols, this protocol addresses the challenge of facilitating the attachment of liver cell lines and primary hepatocytes to glass coverslips for imaging using an inverted fluorescence microscope. Our protocol describes two different loading/labeling strategies for Fura-2 dye: one is cost-effective but requires skillful pipettor handling, and the second one is easy but expensive as it needs a large volume of Krebs-Ringer HEPES (KRH)-Fura-2 solution. If the coverslips are handled properly, the cost-effective coverslip-only loading approach produces similar quality results as the large volume method. Finally, we describe a simple and user-friendly procedure to analyze Ca²⁺ signals over time using Microsoft Excel's functional equations. Key features • This protocol enables real-time monitoring of intracellular Ca²⁺ in living hepatocytes. • The method is based on fluorescence microscopy of hepatocytes loaded with Fura-2 fluorescent dye; moreover, the protocol can be extended to other mammalian cell lines. • We used this protocol to monitor the activity of store-operated Ca²⁺ channels or SERCA pumps, but it could also be extended to other intracellular phenomena. • If the Fura-2 loading strategy and incubation time are followed properly, this protocol produces highly reproducible imaging results.