Fluorescent calcium ion (Ca2+ ) indicator

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.

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.

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.

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

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.[1]

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We report the first demonstration of a fast wavelength‐switchable 340/380 nm light‐emitting diode (LED) illuminator for Fura‐2 ratiometric Ca2+ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura‐2 and enables the precise detection of cytosolic Ca2+ concentrations, which is only limited by the Ca2+ response of Fura‐2. Using this illuminator, we have shown that Fura‐2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca2+ events in tsA‐201 cells and while utilising the 150 μs switching speeds available, it was possible to image spontaneous Ca2+ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca2+ events using Fura‐2.[2]

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.)