Fluorescent calcium ion (Ca2+ ) indicator
Fura-2 AM does not target a specific biological receptor but serves as a fluorescent probe for the detection of intracellular calcium ions (Ca²⁺). Once inside the cell, the AM esters are cleaved by esterases, and the resulting Fura-2 binds to Ca²⁺ with high affinity (Kd ≈ 224 nM at 37°C, pH 7.2) and selectivity. The binding of Ca²⁺ to Fura-2 causes a shift in the excitation spectrum, with the calcium-bound form exhibiting increased fluorescence at 340 nm and decreased fluorescence at 380 nm. The ratio of fluorescence at 340 nm to 380 nm is proportional to the free calcium concentration and can be calibrated using standard calcium buffers. Fura-2 AM does not interfere with cellular calcium signaling pathways, making it a non-invasive tool for studying calcium dynamics. The compound's membrane permeability, ratiometric properties, and high sensitivity make it one of the most widely used calcium indicators for live-cell imaging.

Instruction for use (This is our recommended protocol, which may be adjusted to meet your specific needs).
Fura-2 AM diffuses across the membrane, then de-esterified by cellular esterases to give Fura-2 free acid. [1]
1. In order to prepare 1 mM Fura-2 AM stock solution, add 50 μL of DMSO to a 50 μg vial. It is important to use anhydrous DMSO stored under nitrogen and it is recommended to take DMSO with a needle by puncturing the septum to prevent hydration of the DMSO. After preparing the Fura-2 AM solution keep it in a dark dry place (protect from light). Fura-2 AM in DMSO is stable at ambient temperature for 24 hours and at -20 degrees for several months when stored in a dry container.
2. Aliquot 2 mL of culture media into a 15 mL conical tube, warm to 37°C. and add 2 μL of Fura-2 AM stock solution to obtain a 1μM Fura-2 AM working solution. Vortex the solution vigorously for 1 min.
3. Place the coverslip containing the cells in a 35 mm tissue culture dish along with the loading solution.
4. Incubate the neurons at 37°C for 30 minutes in a dark incubator. Precisely time the incubation.
5. Use a 35 mm dish containing 2 mL of tissue culture media without Fura-2 AM. Remove the coverslip from the loading solution and place in the new dish.
6. Put the coverslip in place within the image chamber.
In vitro studies have demonstrated that Fura-2 AM is a reliable and sensitive indicator of intracellular calcium concentrations. In cell suspensions or adherent cells, Fura-2 AM loading (typically 1-5 µM for 30-60 minutes at 37°C) results in uniform dye distribution within the cytosol. The dye does not accumulate in mitochondria or other organelles, and it is not significantly sequestered by intracellular compartments. The fluorescence signal is stable for several hours after loading, allowing for prolonged imaging experiments. Fura-2 AM has been used to measure calcium responses to a wide range of stimuli, including neurotransmitters, hormones, growth factors, and mechanical stimuli. The compound's ratiometric properties allow for the quantification of absolute calcium concentrations, making it suitable for comparative studies across different cell types and experimental conditions. Fura-2 AM has also been used in high-throughput screening assays to identify modulators of calcium signaling. The compound's loading, calcium binding, and fluorescence properties have been extensively characterized, and it is considered a gold standard for calcium imaging.

In vivo studies using Fura-2 AM are limited, as the compound is primarily used in in vitro and ex vivo applications. However, Fura-2 AM has been used to load cells in vivo in some studies, including the loading of neurons in brain slices and the loading of cells in whole organs for calcium imaging. The compound's membrane permeability allows for the loading of cells in intact tissues, although the limited tissue penetration of UV light and the potential for dye compartmentalization can be challenges. Fura-2 AM has also been used in vivo for the measurement of calcium signals in zebrafish, Drosophila, and other model organisms. However, the compound is not suitable for deep tissue imaging due to the limited penetration of UV light, and other calcium indicators (e.g., genetically encoded calcium indicators, red-shifted dyes) are often preferred for in vivo applications.

Imaging Protocol[1]
1. Calibrate the microscope stage.
2. Load Tyrodes solution into the input line taking care to prevent the formation of air bubbles.
3. Connect the chamber to the perfusion lines and perfuse Tyrodes solution through the chamber once again, taking care to prevent the formation of bubbles in the chamber
4. Place a drop of oil on the objective, place the chamber on the microscope stage and focus on the cells using transmitted light.
5. Examine the fluorescence of the cells using illumination at 340 and at 380 nm using the eyepieces. Resting cells should be dim at 340 and bright at 380. In general, cells should not be illuminated with UV light for more than 10 or 15 seconds and the intensity of the excitation light should be reduced with a neutral density filter to prevent phototoxicity.
6. Examine the cells using the camera and set the gain and exposure of the camera to generate an image that is close to saturation (but not saturated) when illuminated at 380 nm and well below saturation when illuminated at 340 nm. Keep the exposure below 200ms if possible. Once set, do not change the camera gain or exposure unless you plan to also alter the background, RMin and RMax (see below).
7. Collect an image at each wavelength and use the region of interest (ROI) tool to measure the intensity of the background in a variety of locations in the images for each wavelength.
8. Average the background values and enter the background values into the appropriate locations in the imaging program. The background value will be subtracted from each pixel in the field.
9. Collect a practice ratio image and adjust the Threshold values for each wavelength to generate a ratio image that includes only the cells and not the background and that is not noisy in the region close to the edges of the cells.
10. Set the minimum ratio value (RMin) to be about 10% below the lowest ratio of any cell in the field.
11. Set the RMax to be about 12 times the RMin. Do not change the RMin or RMax between experiments that you plan to compare as it will make the comparison difficult. We seldom change the RMin and Rmax values on our imaging rig.
12. Use the automated stage to find the fields that you plan to image, and record the location of each field using the software. We generally collect five fields of view during an experiment. The automated stage moves to a field, collects a ratio image and moves on to the next field.13. Set up the time-lapse interval to collect images between 0.1 and 10 seconds apart depending on the type of signals that you expect to see.
14. Start the experiment.
15. When testing the calcium imaging system it is often useful to use stimuli like high potassium tyrodes (65 mM KCl) or ionomycin (2µM) that will cause an intracellular calcium rise and calcium free extracellular solution that will (containing the calcium buffers like EGTA and BAPTA) that will reduce the calcium concentration.

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Analysis[1]
Once the experiment is complete you will want to convert the set of ratio images into time-lapse calcium measurements for individual cells or regions of interest within cells. To do this:[1]
1. Use the region of interest tool (ROI) to define the areas of the image in which you want to measure calcium. It is generally useful to have at least one ROI that covers the cell body of the cell. When defining the ROIs, it is useful to play the movie of the images to verify that the cells do not move during the course of the experiment.
2. Use the software to collect time-lapse ratio measurements for each ROI in each image.
3. Import the ratio measurements into an analysis program. You will need to view the calcium traces for specific cells or ROIs, average multiple cells or ROIs and convert the ratio measurements to intracellular calcium values. We use a set of macros written in Igor Pro which allow us to do all these common analysis tasks but other programs like Excel, though less convenient can also be used.
4. The equation [Ca] = (R-Rmin)/(Rmax-R)Sf*Kd can be used to convert the Fura-2 ratio values to intracellular calcium concentrations. [Ca] is the calcium concentration, R is the Fura-2 340/380 ratio, RMin and RMax are the 340/380 ratios in the absence of calcium or in the presence of a saturating concentration of calcium respectively; and Sf*Kd is the product of the Kd of Fura-2 (approximately 120 nM at RT) and a scaling value. To measure RMin, RMax and Sf*Kd it is necessary to perform either an in vivo or an in vitro calibration. In vivo calibration requires a combination of patch clamping and calcium imaging, which can be complicated but is also very precise. In vitro calibration can be done using a home-made calibration chamber that consists of two coverslips separated by a thin coverslip spacer to generate a thin layer of solution in which to perform the calibration. The most convenient way of measuring Fura-2 ratio values as a function of calcium concentrations is to use a calibration kit that contain multiple buffered calcium solutions and Fura-2 free acid.
For in vitro loading protocols, Fura-2 AM is typically prepared as a stock solution in DMSO at a concentration of 1-5 mM. The dye is diluted in physiological buffer (e.g., HBSS, Krebs-Ringer buffer) containing 0.02% Pluronic F-127 (a non-ionic detergent that enhances dye solubility) to a final concentration of 1-5 µM. Cells are incubated with the loading solution for 30-60 minutes at 37°C in the dark. After loading, cells are washed with dye-free buffer to remove extracellular dye and allowed to de-esterify for an additional 15-30 minutes at 37°C. The cells are then placed on a microscope stage, and fluorescence imaging is performed using a fluorescence microscope equipped with a UV light source and appropriate filter sets (excitation at 340 nm and 380 nm, emission at 510 nm). Images are acquired using a CCD camera or a confocal microscope, and the ratio of fluorescence intensities at 340 nm and 380 nm is calculated for each cell or region of interest. Calibration of the ratio to absolute calcium concentration is performed using a calcium calibration kit or by measuring the fluorescence ratio at zero calcium (using EGTA) and at saturating calcium (using ionomycin).

Loading of Fura-2 calcium dye[1]
Researchs may load cells with acetoxy-methyl-ester Fura-2 (Fura-2 AM), which diffuses across the cell membrane and is de-esterified by cellular esterases to yield Fura-2 free acid. The exact parameters for Fura-2 loading vary widely across cell types. We recommend testing various conditions by preparing several loading solutions containing a multiple concentrations of Fura-2 raging from 1- 4 µM, incubating cells in the loading solution for a variety of times from 15 minutes to 2 hours and testing the loading at room temperature and at 37 deg. A simplified protocol for cortical neurons is given below:
1. First, prepare the 1 mM Fura-2 AM stock by adding 50µl of DMSO to a 50µg sample-containing vial. It is important to use dry DMSO packed under nitrogen and it is necessary to remove the DMSO with a needle by puncturing the septum to prevent hydration of the DMSO. After preparing the Fura-2 AM solution keep it in a dark dry place. Fura-2 AM in DMSO is stable at RT for 24 hours and is stable at - 20 degrees in a dry container for several months.
2. Aliquot 2 mls of culture media into a 15 ml conical tube, warm to 37 deg. and add 2µl of Fura-2 AM stock to generate a 1µM Fura-2 AM solution. Vortex the solution vigorously for 1 minute.
3. Transfer the loading solution to a 35 mm tissue culture dish and transfer the coverslip with the cells into the dish.
4. Incubate the neurons at 37 degrees for 30 minutes in a dark incubator. Time the incubation precisely.
5. Prepare a 35 mm dish containing 2 mls of tissue culture media without Fura-2 AM. Remove the coverslip from the loading solution and place in the new dish.
6. Mount the coverslip on the imaging chamber. Remove the coverslip from the 35 mm dish and rapidly mount onto the chamber making sure to prevent drying of the cells. We use an imaging chamber manufactured by Warner Instruments that allows a 10mm coverslip containing the cells to be mounted on the bottom and a second coverslip to be mounted on the top forming a sandwich. The two coverslips are secured with vacuum grease to the chamber and two tubes at either end of the chamber allow for perfusion of solutions through the chamber. The input line is connected to a syringe and the output line is connected to a well that is emptied by a suction line connected to a vacuum trap.
For in vitro cell-based assays, Fura-2 AM is used to measure intracellular calcium responses to various stimuli in a wide range of cell types. Cells are seeded on coverslips or in 96-well plates and loaded with Fura-2 AM as described above. The cells are then placed on a microscope stage or in a plate reader, and baseline fluorescence is recorded for 1-5 minutes. The stimulus (e.g., agonist, drug, mechanical stimulation) is added, and fluorescence is recorded for an additional 5-30 minutes. The change in the 340/380 ratio is calculated as a measure of the calcium response. For high-throughput screening, automated plate readers equipped with dual excitation wavelengths are used. All experiments include appropriate positive controls (e.g., ionomycin, ATP, histamine) and negative controls (e.g., vehicle, buffer), and results are expressed as mean ± standard deviation from at least three independent experiments.

For in vivo animal experiments, Fura-2 AM is typically used for ex vivo imaging of tissues (e.g., brain slices, isolated organs) rather than for in vivo imaging. Tissues are incubated with Fura-2 AM (5-10 µM) in oxygenated buffer for 30-60 minutes at 37°C, washed, and then placed on a microscope stage for imaging. Calcium responses to electrical stimulation, neurotransmitter application, or other stimuli are recorded. The compound is not typically administered to live animals for in vivo imaging.

Pharmacokinetic data for Fura-2 AM are not applicable, as the compound is used as a calcium indicator rather than as a therapeutic agent. The compound has a molecular weight of 1001.86 g/mol and a molecular formula of C₄₄H₄₇N₃O₂₄. It is soluble in DMSO and other organic solvents, and it is typically prepared as a stock solution in DMSO. The compound should be stored as a powder at -20°C, protected from light and moisture, and solutions should be used within 1-2 days to prevent hydrolysis of the AM esters.

Fura-2 AM is generally considered to have low toxicity at the concentrations used for calcium imaging (1-5 µM). However, the AM ester groups can be toxic to some cell types at higher concentrations, and the DMSO used as a solvent can also have cytotoxic effects at high concentrations. The compound should be handled with appropriate safety precautions, including the use of personal protective equipment and working in a well-ventilated area. The compound is for research use only and is not intended for human therapeutic use.

[1]. Calcium imaging of cortical neurons using Fura-2 AM. J Vis Exp. 2009 Jan 19;(23):1067.

Calcium imaging is a commonly used technique for measuring calcium signals in cultured cells. Calcium imaging utilizes calcium indicator dyes, which are BAPTA-based organic molecules whose spectral properties change with the binding of Ca²⁺ ions. Calcium indicator dyes are divided into two categories: ratiometric dyes (such as Fura-2 and Indo-1) and single-wavelength dyes (such as Fluo-4). Ratiometric dyes change their excitation or emission spectra with the binding of calcium ions, allowing the determination of intracellular calcium ion concentration by the ratio of fluorescence emission or excitation at different wavelengths. The main advantage of using ratiometric dyes compared to single-wavelength probes is that their ratio signal is independent of dye concentration, light intensity, and optical path length, thus avoiding the influence of these factors and allowing for independent determination of intracellular calcium ion concentration. Fura-2 is one of the most commonly used calcium indicators, with an emission peak at 505 nm that changes from 340 nm to 380 nm with calcium ion binding. This article describes a method for measuring elevated intracellular calcium ion concentrations in neurons and other excitable cells using Fura-2. [1] We have demonstrated for the first time a fast wavelength-switchable 340/380 nm light-emitting diode (LED) illuminator for live-cell Fura-2 ratiometric Ca2+ imaging. The excitation wavelength of this LED is highly matched to the excitation peaks of both bound and free Fura-2, enabling precise detection of cytoplasmic Ca2+ concentrations with accuracy limited only by the Ca2+ response of Fura-2. Using this illuminator, we have demonstrated that concentrations as low as 250 nM of Fura-2 acetoxymethyl ester (AM) can be used to detect induced Ca2+ events in tsA-201 cells. Furthermore, with a switching speed of 150 μs, we were able to image spontaneous Ca2+ transients in hippocampal neurons at a rate of 24.39 Hz, which would be suppressed or disappeared at a typical acquisition rate of 0.5 Hz. In summary, compared with existing systems, the sensitivity and acquisition speed obtained by using this LED illuminator are significantly improved in terms of temporal resolution, and it enables optical imaging of fast Ca2+ events using Fura-2. [2]
Fura-2 AM is a research-use only compound and has not been approved for clinical applications by any regulatory authority. It is also known as Fura-2 acetoxymethyl ester. The compound has a molecular formula of C₄₄H₄₇N₃O₂₄ and a molecular weight of 1001.86 g/mol. Fura-2 AM is a membrane-permeable, UV light-excitable fluorescent calcium indicator that is widely used for the measurement of intracellular calcium concentrations in living cells. The compound is a gold standard for calcium imaging and is used in many areas of biomedical research. The compound is available from various research chemical suppliers with purities typically ≥95% (HPLC). Storage recommendations include keeping the compound in a tightly sealed container, protected from light and moisture, at -20°C.

C44H47N3O24

1001.85

1001.254

108964-32-5

3364574

Light yellow to green yellow liquid

1.4±0.1 g/cm3

975.9±75.0 °C at 760 mmHg

544.0±37.1 °C

0.0±0.3 mmHg at 25°C

1.567

2.9

0

27

37

71

1780

0

O=C(C1=CN=C(C2=CC3=CC(OCCOC4=CC(C)=CC=C4N(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)=C(N(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)C=C3O2)O1)OCOC(C)=O

VPSRLGDRGCKUTK-UHFFFAOYSA-N

InChI=1S/C44H47N3O24/c1-25-7-8-32(46(16-39(53)65-20-60-26(2)48)17-40(54)66-21-61-27(3)49)35(11-25)58-9-10-59-36-12-31-13-37(43-45-15-38(71-43)44(57)69-24-64-30(6)52)70-34(31)14-33(36)47(18-41(55)67-22-62-28(4)50)19-42(56)68-23-63-29(5)51/h7-8,11-15H,9-10,16-24H2,1-6H3

acetyloxymethyl 2-[6-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-1-benzofuran-2-yl]-1,3-oxazole-5-carboxylate

108964-32-5; FURA 2-AM; Fura-2 AM; Bis(acetoxymethyl) 2,2'-((2-(5-((acetoxymethoxy)carbonyl)oxazol-2-yl)-5-(2-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)benzofuran-6-yl)azanediyl)diacetate; Fura-2, AM; MFCD00036976; acetyloxymethyl 2-[6-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-1-benzofuran-2-yl]-1,3-oxazole-5-carboxylate; (acetyloxy)methyl 2-({2-[(acetyloxy)methoxy]-2-oxoethyl}[2-(5-{[(acetyloxy)methoxy]carbonyl}-1,3-oxazol-2-yl)-5-(2-{2-[bis({2-[(acetyloxy)methoxy]-2-oxoethyl})amino]-5-methylphenoxy}ethoxy)-1-benzofuran-6-yl]amino)acetate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)