Fura 2 am

Manufactured by Zeiss

Sourced in Germany

Fura-2 AM is a fluorescent calcium indicator used for measuring intracellular calcium levels in cells. It is a membrane-permeable form of the Fura-2 calcium-binding dye.

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Lab products found in correlation

Fura 2 am, by Thermo Fisher Scientific (5 mentions) Axiovert 200, by Zeiss (2 mentions) Metafluor software, by Molecular Devices (1 mentions) Inverted axioobserver microscope, by Zeiss (1 mentions) Metafluor, by Molecular Devices (1 mentions) Hanks balanced salt solution (hbss), by Zeiss (1 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Axiovert 100, by Zeiss (1 mentions) High speed ccd camera, by Hamamatsu Photonics (1 mentions) Epifluorescence equipped inverted microscope, by Zeiss (1 mentions) Volocity software, by PerkinElmer (1 mentions)

5 protocols using fura 2 am

Intracellular Calcium Dynamics in Co-Cultured PSCs and Pancreatic Cancer Cells

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Calcium imaging was performed according to the previous study^{69 (link)}. To monitor the intracellular calcium concentration, mono-culture and direct co-culture of PSCs and BxPC-3-RFP cells were seeded on 15 mm cover slide in DMEM/F12 medium (supplemented with 2% fetal bovine serum and 10 μg/ml antibiotics/antimycotics) for 48 h. After HBSS wash, cells were loaded with 5 μM Fura-2/AM (Invitrogen) and incubated at 37 °C for 30 min in HBSS (supplemented with Ca²⁺/Mg²⁺, 1% fetal bovine serum, and 10 μg/ml antibiotics/antimycotics). After HBSS wash, cells were incubated for an additional 30 mins at 37 °C in HBSS for esterification of Fura-2/AM and transferred to the recording chamber of an inverted fluorescent microscope (Axiovert 200; Zeiss) with 20× objective lens and MetaFluor software (Molecular Device). Ca²⁺ dependent fluorescence signals were obtained by using excitation at 340 nm and 380 nm, and ratioing the emission fluorescence intensities detected at 500–510 nm. The data of 1 min (60 cycles) were collected for calculation. From this ratio, the level of intracellular Ca²⁺ can be estimated.

Chen Y.I., Chang C.C., Hsu M.F., Jeng Y.M., Tien Y.W., Chang M.C., Chang Y.T., Hu C.M, & Lee W.H. (2022). Homophilic ATP1A1 binding induces activin A secretion to promote EMT of tumor cells and myofibroblast activation. Nature Communications, 13, 2945.

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Fura-2AM Calcium Imaging in Cortical/Hippocampal Neurons

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Mouse cortical/hippocampal neurons (19–23 DIV) were loaded in neurobasal containing 20 μm Fura-2AM (Invitrogen) for 30 min. After two washes in physiological 1.6 mM calcium-containing buffer (139 mM NaCl, 1.25 mM glucose, 15 mM Na₂HPO₄, 1.8 mM MgSO4, 1.6 mM CaCl₂, 3 mM KCl, 10 mM HEPES), Fura-2AM-loaded neurons were imaged at 37 °C on an inverted AxioObserver microscope (Carl Zeiss) equipped with a 300 W Xenon lamp (Suttler instruments) and a Fluar 40× (numerical aperture (NA) 1.4) oil immersion objective. Fura-2AM was sequentially excited at 340 and 380 nm and the emission monitored at 510 nm. Images were acquired with a cascade 512 EMCCD camera every 2 s and digitized using Metafluor software (Roper scientific). The intracellular calcium concentration was estimated by measuring the F340/380 nm ratio of fluorescence. Neurons were treated for 40 s with 100 μM DHPG in 1.6 mM calcium-containing buffer.

Khayachi A., Gwizdek C., Poupon G., Alcor D., Chafai M., Cassé F., Maurin T., Prieto M., Folci A., De Graeve F., Castagnola S., Gautier R., Schorova L., Loriol C., Pronot M., Besse F., Brau F., Deval E., Bardoni B, & Martin S. (2018). Sumoylation regulates FMRP-mediated dendritic spine elimination and maturation. Nature Communications, 9, 757.

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Calcium Imaging of Motor Neurons

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MNs were dissociated on differentiation day 5, plated on coated 22 X 22 mm glass coverslips, and grown in MN medium until calcium imaging experiments. HEK293T cells were cultured on 0.1% gelatin-coated 22 X 22 mm glass coverslips and transfected with plasmid DNAs for 24 h before calcium imaging. Before calcium imaging, the MNs were treated with 2uM Fura-2-AM (Invitrogen) in HBSS (Invitrogen) containing 2 mM CaCl₂ in the dark chamber and incubated at 37 °C, 5% CO₂ for 45 min. The excess Fura-2-AM was washed out with HBSS (without CaCl₂) and incubated for an additional 30 min in HBSS (containing 2 mM CaCl₂) for recovering, and then the coverslips were transferred onto the recording chamber of an inverted fluorescence microscope (Zeiss Axiovert 200) equipped with a 20 X objective lens and MetaFluor (Molecular Devices) acquisition and analysis software. The fluorescence signals at 510 nm were acquired every 2 s in 5 min by UV excitation at wavelengths of 340 nm (indicating calcium ion-bound- Fura-2-AM) and 380 nm (indicating calcium ion-free-Fura-2-AM), respectively. The formula

R = \frac{Δ 340}{Δ 380}

, in which the ∆ indicated the values of 340 nm or 380 nm minus their background values, was used to calculate and compare the relative levels of intracellular calcium ion of different types of MN in culture.

Huang S.L., Wu L.S., Lee M., Chang C.W., Cheng W.C., Fang Y.S., Chen Y.R., Cheng P.L, & Shen C.K. (2020). A robust TDP-43 knock-in mouse model of ALS. Acta Neuropathologica Communications, 8, 3.

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Fura-2 AM-based Ca2+ Imaging Protocol

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For Ca²⁺-imaging experiments, the cells grown on glass coverslips were loaded for 20–30 min with 5 μM 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(21-amino-51-methylphenoxy)-ethane-N,N,N1,N1-tetraacetic acid penta-acetoxymethyl ester (Fura-2 AM; LifeTechnologies) in control buffer medium containing (in mM): 140 NaCl, 3 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, pH 7.4 with NaOH. At the end of the Fura-2 AM-loading period, the coverslip was introduced into a microscope chamber on the stage of an inverted microscope (Axiovert 100, Carl Zeiss, Jena, Germany) equipped with a 20x objective (Neofluar, Carl Zeiss). Cells were washed twice with the control buffer medium, and maintained in the same solution throughout the experiment. Fluorescence measurements were performed at room temperature with a combination of a monochromator (Polychrome II, TILL Photonics, Gräfelfing, Germany) and a cooled CCD camera (IMAGO, pco, Kelheim, Germany) operated with TILLvisION (TILL Photonics) software. Pairs of images (excitation at 340 and 380 nm) of Fura-2 fluorescence intensity were measured every 1 s; background fluorescence was subtracted in each experiment. Fluorescence values were converted to [Ca²⁺]_i using a calibration curve, as described (Grynkiewicz et al., 1985 (link)).

Mancini M., Soldovieri M.V., Gessner G., Wissuwa B., Barrese V., Boscia F., Secondo A., Miceli F., Franco C., Ambrosino P., Canzoniero L.M., Bauer M., Hoshi T., Heinemann S.H, & Taglialatela M. (2014). CRITICAL ROLE OF LARGE CONDUCTANCE VOLTAGE- AND CALCIUM-ACTIVATED POTASSIUM CHANNELS IN LEPTIN-INDUCED NEUROPROTECTION OF N-METHYL-D-ASPARTATE-EXPOSED CORTICAL NEURONS. Pharmacological research : the official journal of the Italian Pharmacological Society, 0, 80-86.

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Intracellular Calcium Measurement in Myotubes

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Intracellular calcium [Ca²⁺]i was measured in day-6 myotubes as previously described⁶⁴. Myotubes on coverslips were loaded with 5 mM Fura-2 AM (Invitrogen) in 1x regular rodent Ringer’s solution (RRS) at RT for 45 min. For measurement in heat and caffeine status, myotubes were incubated with 50 μM 4CmC at 37 °C for 10 min just after loaded Fura-2 AM, and then mounted in a chamber set at 37 °C on the stage of an epifluorescence-equipped inverted microscope (Zeiss). Myotubes were sequentially excited at 340 and 380 nm wavelength and fluorescence emission at 510 nm was collected using a high-speed CCD camera (Hamamatsu). The ratio of the fluorescence intensity (R340/380) was analyzed using Volocity Software (PerkinElmer). For calculating the intracellular calcium concentration, the maximum ratio (Rmax) and minimum ratio (Rmin) were determined by using ionomycin (20 μM) in RRS followed by EGTA solution (125 mM) at the end of each experiment. [Ca²⁺]i of each myotube was calculated as previously described^{65 (link)}.

Endo Y., Groom L., Celik A., Kraeva N., Lee C.S., Jung S.Y., Gardner L., Shaw M.A., Hamilton S.L., Hopkins P.M., Dirksen R.T., Riazi S, & Dowling J.J. (2022). Variants in ASPH cause exertional heat illness and are associated with malignant hyperthermia susceptibility. Nature Communications, 13, 3403.

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