Fura-2 AM is a widely used ratiometric fluorescent dye for measuring intracellular calcium levels in live cells. This protocol describes how Fura-2 AM imaging is performed, detailing the step-by-step execution of reagent preparation, cell loading, and calcium imaging. It is optimized for various cell types, including fibroblasts, PC12 cells, and embryonic neurons. The procedure includes buffer preparation, cell replating, dye loading, and imaging setup. Designed for reproducibility and sensitivity, this protocol enables researchers to monitor calcium dynamics with high temporal resolution. It is compatible with standard fluorescence microscopy and microplate readers, making it suitable for both single-cell and high-throughput applications.

Introduction

Calcium ions play a pivotal role in cell signaling, influencing processes such as neurotransmission, muscle contraction, and hormone secretion. Fura-2 AM is a cell-permeable, high-affinity calcium indicator that allows researchers to visualize and quantify intracellular calcium fluctuations. This protocol provides a reliable method for using Fura-2 AM in live-cell imaging experiments. By leveraging its ratiometric properties, the protocol minimizes variability due to dye loading and photobleaching. Whether you are studying GPCR activation or calcium channel dynamics, this guide ensures accurate and reproducible results across a range of cell types and experimental conditions.

Background and principles

Fura-2 AM is a synthetic, esterified form of the Fura-2 dye that can permeate cell membranes. Fura-2 AM was developed to improve the accuracy and usability of calcium imaging. Once inside, cellular esterases cleave the AM groups, trapping the active dye intracellularly. The dye binds calcium ions by forming a complex, which changes its fluorescence properties. The dye exhibits calcium-dependent shifts in excitation wavelengths: 340 nm when bound to calcium and 380 nm when unbound, while maintaining a stable emission at 510 nm. This ratiometric property allows for precise quantification of calcium concentrations, independent of dye concentration or cell thickness. The protocol utilizes HBSS buffer and BSA to facilitate dye loading and minimize background fluorescence, ensuring optimal signal-to-noise ratios during imaging.

Stage 1 - Reagent preparation

Materials required

• Fura-2 AM (for example ab120873)
• HBSS-1X (see recipe below)
• 50 mM KCl (see recipe below)

Steps

Mix Fura-2 AM with DMSO.

• Add 50 µL of DMSO to 50 µg of lyophilized Fura-2 AM and vortex for 1 min.

Prepare Hank’s Buffered Salt Solution 10X (HBSS-10X).

• For 1000 mL, use 850 mL of distilled H₂0 (18 MΩ) and add the reagents from the table below, stirring.
• Bring to 1000 mL final volume.
• Store at 4°C.

Prepare HBSS-1x solution.

• Mix 100 mL of the 10X HBSS solution (prepared in the previous step) with 800 mL of distilled H₂O.
• Add 0.14 g of anhydrous CaCl₂ (FW 111, 1.3 mM), 1g of d-glucose (FW 180.2, 5.5 mM), and 0.35 g NaHCO₃ (FW 84.01, 4.2 mM).
• Bring to 1000 mL volume with distilled H₂O, pH to 7.4, and store for ~1 week at 4°C.

Prepare 50mM KCl solution.

• 1 liter volume, pH 7.35, ~290-310 mOsm. Store 4°C.

Stage 2 - Replating

Steps

One to two days prior to experimentation, replate cells to collagen coated coverslips (round, glass, sterilized with ethanol, dried) placed in 35 mm tissue culture dishes.

Place 35 mm dishes in 150 mm Petri dishes that will be used as a microincubator and carrier between incubator and experiments.

When replating, place cells in center of round glass coverslips to settle and ensure that imaging is optimized.

Empirically determine the density of cells and how to replate to have cells stick to glass through the washes and the perfusion of solutions.

Stage 3 - On the day of FURA-2 AM imaging

Materials required

• Fura-2 AM (for example ab120873)
• Ionomycin, free acid (for example ab120370)
• HBSS-1X

Steps

Take the HBSS and 50 mM K solutions out of the refrigerator, turn on equipment and perform calibration curve.

• Check that cells are well adhered to the glass.

Take two 50 mL falcon tubes and label as HBSS and HBSS+BSA.

• Pour HBSS from the HBSS container into the 50 mL tube labeled as HBSS.

Prepare HBSS+BSA solution.

• Measure ~45 mg of BSA (Fatty acid free) and add to the empty tube labeled HBSS+BSA.
• Transfer ~45 mL of HBSS to the same tube and mix gently so as not to create a lot of bubbles, but to mix the BSA completely, then let it settle.
• This should be a 1 mg/mL mixture of BSA and HBSS.

Mix Fura-2 and HBSS+BSA.

• Thaw the Fura-2 AM in 50 µl DMSO and mix with room lights turned off.
• Only load 2 of the 35 mm dishes at a time, as the timing for imaging will not work well for the timing of the post-Fura wash.
• Pipette 4-10 µL/35 mm dish of cells of this Fura-2/HBSS/BSA mixture into a 15 mL Falcon tube (a lower concentration of Fura-2 AM in the cells for imaging will reduce the intracellular buffering capacity).
• Add 4 mL of HBSS+BSA into the 15 mL tube with Fura-2.
• Get two dishes of coverslips from the incubator, place them on the bench, then vortex the 15 mL tube on high for 1 min.
• Set in a rack with the 2 x 50 mL tubes of HBSS and HBSS+BSA (loosen/remove lids to all Falcon tubes).

Load Fura.

• Remove 2 of the 35 mm dishes from the incubator.
• With 1 mL sterile pipette tips, remove all media from one 35 mm dish of cells and eject it into a waste container.
• With the same tip, bring up 1 mL of HBSS and gently add to the cells, being careful to place along the side and gently not to cause disruption of plated cells.
• Pull up the same 1 mL of HBSS and eject it into the waste container, bringing up the next 1 mL of HBSS with the same tip and placing gently on the cells.
• Remove the solution again and discard the tip.
• With a new sterile pipette tip, pipette up 1 mL of HBSS+BSA and wash gently, removing waste.
• With the same pipette tip, repeat two more times to wash the cells 3x with HBSS + BSA.
• With the same tip, immediately add 1 mL of the Fura +HBSS+BSA that was vortexed for 1 min.
• Add the second 1 mL of Fura-2 AM to the cells and label the lid of the coverslip dish with the time.  Repeat for the second dish.
• Typically, load for 45 min, but this might need to be varied as well as the 4-10 µL of Fura-2 AM in 2 mL of HBSS+BSA. Replace both dishes in the CO₂/37°C incubator for the 45 min loading time.

Wash the cells.

• Remove both dishes and gently, with a new sterile tip, pull 1 mL of the Fura-HBSS+BSA mix to waste and then the second 1 mL.
• With a new pipette tip, wash the cells 3-4 times with 1 mL of HBSS (not HBSS+BSA) each time gently, and place the wash in the waste.
• After the 4^th wash, leave on 1 mL of HBSS and add a second mL of HBSS.  Label the time and replace it in the incubator for 30 min-45 min.

Perform imaging.

• Get the first coverslip of cells about 25 min into the wash and set up on the imaging rig in the chamber.
• Perform the imaging experiment in 7-15 min maximum, and then get the next dish from the incubator.
• Constantly perfuse the cells with HBSS on the recording chamber for resting Ca²⁺ measurements. To stimulate cells, use a stimulant of some sort, either the test solution, or 50 mM K solution, or electrical stimulation.
• To stimulate, use a range of high K⁺ solutions (matched monovalents): 20 mM, 40 mM and 60 mM KCl solutions as well, each time replacing NaCl with equivalent amounts of KCl.
• To obtain a positive control for imaging, use 20 µM working concentration ionomycin (ab120370, 5 mg free acid mixed to 20 mM in DMSO) by placing the appropriate volume into the volume of the chamber while perfusing.

To continue through the day, we recommend beginning the next two coverslips of cells loading about the same time as washing the first sets.

• Keep track of loading/washing, and know how long it takes to switch coverslips and do the imaging throughout so you can keep to time.

Choosing the right indicator

Selecting the optimal calcium indicator is a foundational step for achieving accurate measurement of intracellular calcium concentrations. With a variety of calcium indicators available, such as Fura-2, Fluo-4, and Indo-1, it is important to consider the specific requirements of your experiment, including the type of cells being studied, the desired emission wavelengths, and the sensitivity needed for detecting calcium binding events. Ratiometric calcium indicators like Fura-2 are particularly advantageous for experiments where uneven loading, dye leakage, or photobleaching could compromise data quality. These indicators provide a ratiometric readout, allowing for more reliable measurement of calcium concentrations across cells of varying thickness and morphology. In contrast, non-ratiometric indicators such as Fluo-4 offer a simpler workflow but may require additional calibration to account for differences in dye loading and cell volume. By carefully matching the calcium indicator to your experimental design, you can ensure precise and reproducible measurement of intracellular calcium levels, leading to more meaningful insights into cellular calcium dynamics.

Indicator properties

Understanding the properties of calcium indicators is essential for optimizing calcium imaging experiments and ensuring accurate measurement of intracellular calcium concentrations. Fura-2, for example, is renowned for its greatly improved fluorescence properties compared to earlier dyes like Quin 2, enabling researchers to conduct experiments at lower indicator concentrations while maintaining high sensitivity. The emission spectra of calcium indicators, such as Fluo-4, allow for the detection of subtle changes in calcium levels within living cells. Ratiometric indicators like Fura-2 provide an additional layer of accuracy by enabling ratio measurements at two excitation wavelengths (340 nm and 380 nm), which helps correct for variations in dye concentration, cell thickness, and photobleaching. This built-in calibration ensures that changes in fluorescence emission directly reflect changes in intracellular calcium concentrations. By selecting indicators with the right spectral and binding properties, researchers can tailor their protocols for specific cell types and experimental conditions, ultimately achieving more accurate and reproducible results.

Spectral characteristics

The spectral characteristics of calcium indicators are central to their effectiveness in detecting changes in intracellular calcium concentrations. Each indicator has unique excitation and emission spectra that shift in response to calcium binding, providing a sensitive means to monitor calcium levels in real time. For example, Fura-2 exhibits a calcium-dependent shift in excitation wavelengths, exciting at 340 nm when bound to calcium and at 380 nm when unbound, while maintaining a consistent emission at 510 nm. This ratiometric approach allows for accurate measurement of calcium concentrations by comparing fluorescence emission at these two excitation wavelengths, effectively compensating for factors such as uneven dye loading or cell thickness. Other indicators, like Fluo-4, rely on changes in emission spectra to signal calcium binding events. By understanding and leveraging the spectral properties of calcium indicators, researchers can optimize their imaging protocols, select appropriate filter sets, and ensure that their measurements of intracellular calcium concentrations are both sensitive and specific to the cellular events of interest.

Comparison to other methods

Compared to single-wavelength dyes like Fluo-4 or Calbryte 520, Fura-2 AM offers superior accuracy through ratiometric measurement, reducing artifacts from uneven dye loading and photobleaching. Unlike genetically encoded calcium indicators (GECIs), Fura-2 AM does not require transfection, making it ideal for primary cells and short-term experiments. While GECIs provide long-term tracking, Fura-2 AM excels in acute assays with rapid calcium flux. Additionally, the no-wash variant of the Fura-2 assay simplifies workflows for high-throughput screening.

Applications

Fura-2 AM imaging is used extensively in neuroscience, cardiology, and pharmacology to study calcium signaling. It enables real-time monitoring of calcium influx in response to stimuli such as neurotransmitters, ionophores, or electrical pulses. The protocol supports applications in GPCR screening, synaptic activity analysis, and calcium channel characterization. It is also suitable for evaluating drug effects on calcium homeostasis in various cell lines. With compatibility for confocal microscopy and microplate readers, Fura-2 AM is a versatile tool for both qualitative and quantitative calcium imaging in live cells.

Limitations

Despite its advantages, Fura-2 AM imaging has limitations. The dye’s sensitivity to light requires imaging in dark conditions, and its intracellular buffering capacity can affect calcium dynamics if overloaded. The protocol demands precise timing and handling to ensure consistent dye loading and wash steps. Additionally, the need for multiple washes and incubation periods may limit throughput. Fura-2 AM is not ideal for long-term imaging due to dye leakage and photobleaching. For chronic studies or in vivo applications, genetically encoded indicators may be more suitable.

Troubleshooting

Common issues in Fura-2 AM imaging include poor dye loading, high background fluorescence, and inconsistent calcium signals. To improve loading, ensure cells are well adhered and use freshly prepared HBSS+BSA buffer. Avoid creating bubbles during mixing and maintain consistent incubation times. If fluorescence is weak, verify the dye concentration and check for light exposure during preparation. An uneven signal may result from cell detachment; optimize replating density and washing technique. For calibration errors, confirm equipment settings and perform a standard curve using ionomycin. Always conduct imaging in the dark to preserve dye integrity.

Data analysis and interpretation

Accurate data analysis and interpretation are critical for extracting meaningful information from calcium imaging experiments using molecular probes like Fura-2. Ratio measurements, a hallmark of ratiometric calcium indicators, require careful calibration to translate fluorescence ratios into precise intracellular calcium concentrations. Calibration typically involves using known calcium buffers and ionophores to generate standard curves, ensuring that ratio values correspond to actual calcium levels within the cell. When analyzing data, it’s important to account for potential artifacts such as uneven loading, dye leakage, and the effects of photobleaching, all of which can impact the reliability of calcium concentration measurements. Advanced imaging applications and flow cytometry techniques, combined with modern data analysis software, have greatly improved the accuracy and reproducibility of these measurements. By following best practices in calibration, data acquisition, and analysis, researchers can confidently interpret calcium signals, uncovering the complex relationships between intracellular calcium dynamics and cellular function across a wide range of cell types and experimental conditions.