Ratiometric calcium imaging was performed on neurons to detect intracellular calcium levels, as we have previously described (48 (link)). Briefly, cortical neurons on coverslips in 24 well plates were treated with Fura-2-AM fluorescence dye (molecular probes, Life Technologies, USA) in a final concentration of 5 μl/ml and incubated at 37 °C with 5% CO2 for 20 min. After rinsing twice with 37 °C PBS Mg2+-free buffer, the coverslips were transferred into a new 35 mm dish containing 3 ml PBS Mg2+-free buffer. Fura-2 fluorescence was measured at 510 nm emission with 340/380 nm dual excitation selected by a DG-5 system (Sutter Instrument Company, Novato, CA) and imaged with a fluorescence microscope (Leica DMI4000B, Germany). The intracellular calcium ([Ca2+]i) concentration was expressed as ratios from fluorescence intensity between the two excitation wavelengths of R340/380 of Fura-2. Mg2+-free buffer containing 45 mM KCl was applied to cells to induce [Ca2+]i in order to confirm neuronal membrane potential and viability following all treatments. All measurements were independently repeated at least three times. For each experiment, at least 10 cells were selected for analysis.
Fura 2 am fluorescence dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fura-2-AM is a fluorescence dye used in research applications to measure intracellular calcium levels. It is a cell-permeable form of the Fura-2 calcium indicator that can be loaded into cells. Upon binding to calcium, Fura-2-AM exhibits a shift in its excitation spectrum, allowing for ratiometric measurement of calcium concentration.
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4 protocols using fura 2 am fluorescence dye
1
Ratiometric Calcium Imaging of Neurons
2
Ratiometric Calcium Imaging of Neurons
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Ratiometric calcium imaging was performed on neurons to detect intracellular calcium level as we have previously described 29 . Briefly, cortical neurons on coverslips in 24 well plates were treated with Fura-2-AM fluorescence dye (molecular probes®, Life Technologies, USA) in a final concentration of 5 µL/mL, and incubated at 37ºC with 5% CO2 for 20 min.
After rinsing twice with 37 °C PSS Mg 2+ -free buffer, the coverslips were transferred into a new 35 mm dish containing 3 mL PSS Mg 2+ -free buffer. Fura-2 fluorescence was measured at 510 nm emission with 340/380 nm dual excitation selected by a DG-5 system (Sutter Instrument Company, Novato, CA) and imaged with fluorescence microscope (Leica DMI4000B, Germany). The intracellular calcium ([Ca 2+ ]i) concentration was expressed as ratios from fluorescence intensity between the two excitation wavelengths of R340/380 of Fura-2. Mg 2+ -free buffer containing 45 mM KCl was applied to neurons to induce [Ca 2+ ]i in order to confirm neuronal membrane potential and viability following all treatments. All measurements were independently repeated for at least 3 times. For each experiment, at least 30 neurons were selected for analysis.
After rinsing twice with 37 °C PSS Mg 2+ -free buffer, the coverslips were transferred into a new 35 mm dish containing 3 mL PSS Mg 2+ -free buffer. Fura-2 fluorescence was measured at 510 nm emission with 340/380 nm dual excitation selected by a DG-5 system (Sutter Instrument Company, Novato, CA) and imaged with fluorescence microscope (Leica DMI4000B, Germany). The intracellular calcium ([Ca 2+ ]i) concentration was expressed as ratios from fluorescence intensity between the two excitation wavelengths of R340/380 of Fura-2. Mg 2+ -free buffer containing 45 mM KCl was applied to neurons to induce [Ca 2+ ]i in order to confirm neuronal membrane potential and viability following all treatments. All measurements were independently repeated for at least 3 times. For each experiment, at least 30 neurons were selected for analysis.
3
Chemokine-induced Calcium Signaling Assay
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The procedure has been described previously (Nakayama et al., 2010 (link)). Briefly, cells were loaded with 3 μM fura 2-AM fluorescence dye (Thermo Fisher Scientific). After washing, the cells were placed on a F3000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) and stimulated with each recombinant human chemokine. Emission fluorescence at 510 nm was measured upon excitation at 340 and 380 nm, and the fluorescence intensity ratio (R340/380) was obtained.
4
Intracellular Calcium Imaging with Fura-2
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The procedure has been described previously (18 (link)). Briefly, cells were loaded with 3 μM fura 2-AM fluorescence dye (Thermo Fisher Scientific). After washing, cells were placed on F3000 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan) and stimulated with recombinant chemokines. Emission fluorescence at 510 nm was measured upon excitation at 340 and 380 nm, and the fluorescence intensity ratio (R340/380) was obtained.
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