Background

Intracellular calcium plays a central role in regulating key cellular processes, including transcription, signal transduction, excitability, motility, and apoptosis in hepatocytes [1,2]. The concentration of free ionized Ca²⁺ in the cytoplasmic space of hepatocytes is approximately 10^-7 M (0.1 μM) at rest, whereas a large proportion (99%) of total Ca²⁺ in the cytoplasmic space is bound to proteins and metabolites [3]. Ca²⁺ is stored (accumulated) in the endoplasmic reticulum (ER), sarcoplasmic reticulum, mitochondria, and other intracellular organelles. For example, the total Ca²⁺ concentration in the ER can be 10 mM, with a free Ca²⁺ concentration of 10 μM. Hormones, neurotransmitters, and other extracellular signals increase cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) from 0.1 to 1–5 μM [4]. Since the hormonal and metabolic cues are associated with dynamic fluctuations of intracellular Ca²⁺, accurate quantification of cytosolic Ca²⁺ dynamics has been central to interpreting liver-specific signaling pathways. Intracellular fluorescent Ca²⁺ reporters provide one of the best techniques to measure the free Ca²⁺ concentration in the cytoplasmic space and organelles. Fura-2 acetoxymethyl ester (Fura-2 AM), which enables calibrated measurements of absolute intracellular Ca²⁺ concentrations in isolated cells, is one of the most widely used fluorescent indicators for intracellular Ca²⁺ imaging.

With a variety of calcium indicators available, such as fluorescence dyes including Fluo-3, Fluo-4, Fura-2, and Indo-1, or genetically encoded calcium indicators (e.g., GCaMP), it is important to consider the specific requirements of the experiment, including the type of cells being investigated, the desired emission wavelengths, and the sensitivity needed for detecting calcium binding events. Since chemical Ca²⁺ indicators are not required to be transfected or expressed into cells, they provide an unparalleled flexibility in experimental design for tracking intracellular calcium dynamics, largely outperforming genetically encoded indicators in terms of commercial availability and customization. The main advantage of using ratiometric dyes (e.g., Fura-2) compared to single-wavelength probes (e.g., Fluo-3) is that the ratio signal remains unchanged by variations in dye concentration, optical path length, and/or light intensity. This property of ratiometric dyes permits investigators to determine the intracellular calcium concentration with greater accuracy. The primary advantage of Fura-2 over other calcium reporters is its ratiometric capability, which calculates calcium concentration by taking the ratio of fluorescence emitted at 340 and 380 nm excitation [5–7]. This mathematically cancels out experimental artifacts like uneven dye loading, cell thickness variations, and photobleaching, allowing for absolute calcium quantification [4]. While both Fura-2 and Indo-1 are ratiometric indicators, Fura-2 remains the superior choice for fluorescence imaging because of its high resistance to photobleaching compared to the Indo-1 reporter [8]. The principle for the measurement of cytoplasmic Ca²⁺ using this Ca²⁺ fluorescent dye is that the acetoxymethyl ester (AM) form of the fluorescent dyes can diffuse passively across the cell membrane (and intracellular membranes); once the dye is inside the cell, esterases cleave off the AM groups [5,6]. Intracellular or cytoplasmic Ca²⁺ binds to Fura-2 free acid and provides fluorescence intensity (Figure 1).

Figure 1. Principles of Fura-2 AM calcium imaging. Once the Fura-2 AM dye is inside the cell, intracellular esterases cleave the AM groups [5,6]. Intracellular or cytoplasmic Ca²⁺ can bind to Fura-2 free acid, providing fluorescence intensity.

One important methodological gap in existing Fura-2 imaging protocols and tutorials is the lack of a framework for relating image-based measurements to quantitative interpretation. The Grynkiewicz formula (for details, see section E of this protocol) is used to accurately calculate intracellular free Ca²⁺ concentrations using ratiometric fluorescent indicators like Fura-2 [9]. Eventually, the formula translates background-corrected fluorescence ratios into a reliable Ca²⁺ concentration. In this protocol, to successfully use the formula in calibration-based analysis, we have followed a specific protocol (ionomycin-EGTA assay) to acquire the values of different variables and parameters used in the formula. In the case of the image-based implementation workflow, before calculating the ratio, we have subtracted background noise (from optics or autofluorescence) for each excitation wavelength to prevent large calculation errors. With the imaging software, we also computed the pixel-by-pixel intensity ratio 340 nm/380 nm across the entire image through the selection of 10–15 regions of interest. This ratiometric approach inherently corrects for uneven dye loading, minor dye leakage, and photobleaching. Finally, we performed the calibration assay to convert the Fura-2 dye fluorescence ratio into the cytoplasmic Ca²⁺ concentration.

Our group used Fura-2 dye to examine Ca²⁺ signaling by fluorescence microscopy in hepatocytes isolated from Alms1 mutant mice [10], hepatocytes from obese Zucker rats and Hooded Wistar rats [11], and H4IIE rat liver cell lines [12]. Hepatocytes can be easily isolated and purified from rat or mouse liver tissue. Freshly isolated primary hepatocytes, however, only survive for a few hours in non-adherent suspension culture [13,14] and are not amenable to attaching onto commercially available coverslips. This protocol addresses the well-known challenge of cell adhesion by employing an acid (hydrochloric acid, 1 M) washing strategy. As attachment-dependent cells, acid washing provides conditions on the coverslips in which primary hepatocytes can survive. Hydrochloric acid (1 M) washing removes microscopic debris, dust, and toxic manufacturing residues from the glass surface of coverslips, ensuring a chemically sterile environment. The acid treatment hydroxylates the glass surface and yields a highly charged, hydrophilic surface that facilitates robust bonding of extracellular matrix (ECM) proteins of hepatocytes [15]. From our experience, if the acid washing protocol for coverslip is appropriately followed, freshly isolated hepatocytes are shown to adhere and survive up to 36–40 h. It is worth noting that the acid washing strategy is reasonably cost-effective compared to the collagen- or Polylysine-coating method. Furthermore, in this protocol, we have adopted a new and cost-effective Fura-2 dye loading approach, which produces similar quality results as the traditional method using a large dye volume.

The detailed step-by-step protocol described below, using chemical Ca²⁺ indicators, can also be readily adapted to various suspensions and other adherent cell types.