Calcium-FURA2 imaging

Fura-2 is a UV light–excitable, ratiometric Ca2+ indicator and became most common dye of choice for ratio-imaging microscopy, where it is more practical to change excitation wavelengths than emission wavelengths. Upon binding Ca2+, Fura 2 exhibits an absorption shift that can be observed by scanning the excitation spectrum between 300 and 400 nm, while monitoring the emission at ~510 nm. Fura 2 shows comparably good photostability

Storage:  Ambient Temperature
Shipping Condition:  Ambient Temperature
Chemical Name: 1-[6-Amino-2-(5-carboxy-2-oxazolyl)-5 - benzofuranyloxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid, penta-potassium salt
CAS Number: 11364-64-7, 96314-98-6

Fura-2 is a widely used UV-excitable fluorescent calcium indicator. Upon calcium binding, the fluorescent excitation maximum of the indicator undergoes a blue shift from 363 nm (Ca2+-free) to 335 nm (Ca2+-saturated), while the fluorescence emission maximum is relatively unchanged at ~510 nm.

Fura-2 excitation spectra: calcium-free peak at ~380 nm shifts to ~340 nm upon Ca²⁺ binding; emission monitored at ~510 nm.

Further Information on fura-2

Fura 2 was developed to improve the fluorescent properties of Quin 2. The signal intensity in 1 mM of loaded Fura 2 corresponds to that of 30 mM of loaded Quin 2. This characteristic allows an experiment at a lower concentration of indicator using Fura-2 as compared to Quin 2. Fura 2 is one of the most widely used calcium indicators for ratiometric measurement. As a result, many types of instrumentation are now available for experiments using Fura 2. Fura-2 is highly suitable for digital imaging microscopy. It is less susceptible to photo-bleaching than Indo 1. Changes in the cell shape can sometimes affect the fluorescent ratio at 340 nm and 380 nm. For example, fluorescent signal intensities at these wavelengths sometimes decrease simultaneously with smooth muscle contraction. For blood vessels, however, the increase of the signal intensity at 340 nm tends to be smaller on contraction, while the decrease of the signal intensity at 380 nm tends to be larger with its contraction. Fura 2-AM is an acetoxymethyl ester derivative of Fura 2 that can be easily loaded into cells by incubation.

General fura-2 Protocol

General Protocol (for NG 108-15/ Neuronal Cell Line) Reagents:

• 1 mM Fura 2-AM/DMSO (1 mg Fura 2-AM in 1 ml DMSO)
• Hanks·balanced salt solution (HBSS)
• HEPES buffer saline (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,
• 0.8 mM MgCl2, 13.8 mM glucose, pH 7.4)

Protocol:

1. Culture cells on a glass-bottom dish using DMEM containing 5% fetal calf serum.
2. Change the medium to 1 mM dibutyl cAMP/DMEM, and culture the cells for 3-4 days to induce dendrites.
3. Dilute 1 mM Fura 2-AM DMSO solution with HEPES buffer saline to prepare 1 mM Fura 2-AM working solution.
4. Remove the culture medium, and add 0.5 ml of the Fura 2-AM working solution to the cells.
5. Incubate for 20 min. Then remove the Fura 2-AM working solution.
6. Wash the cells once with HEPES buffer saline. Then incubate the cells for 1 hour in the HEPES buffer saline.
7. Use the cells for fluorescent calcium ion detection.
8. Monitor the excitation spectra at 380 nm (calcium free) and 340 nm (calcium complex) with fixed emission at 510 nm. Cell staining conditions differ by cell types, so it is necessary to optimize the conditions for each experiment.

Notes on CALM setup

• We have found on our Timelapse Microscope the 380nm LED is folds brighter that the 340nm LED. Ask us for help to make sure you know how to turn down the power on the LED to be able to detect the ratio change between 380nm and 340nm.
• The 510nm emission filter is currently in the Fura Dichroic Cube