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TRPML Subfamily of Endolysosomal Channels
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Nicholas E. Karagas, Morgan A. Rousseau, Kartik Venkatachalam
Fura-2 is maximally excited at 340 nm when bound to Ca2+ or 380 nm when unbound, and always emits maximally at 510 nm (Paredes et al., 2008). Therefore, the ratio of fura-2 emission at 510 nm following excitation at 340 nm and 380 nm, respectively, is a function of cytosolic free [Ca2+]. Since fura-2 measurements are inherently ratiometric, potential sources of error such as varying concentration of dye in individual cells are of limited concern.
Biochemical Methods of Studying Hepatotoxicity
Published in Robert G. Meeks, Steadman D. Harrison, Richard J. Bull, Hepatotoxicology, 2020
Prasada Rao S. Kodavanti, Harihara M. Mehendale
Currently, the most popular method for measuring free-Ca2+ in mammalian cells is to monitor the fluorescence of Ca2+ indicators like Quin-2 or Fura-2 (Tsien, 1980, 1983; Tsien et al., 1982; Grynkiewicz et al., 1985). These calcium indicators like Fura-2 are loaded into intact cells by incubating with a membrane-permeant acetoxymethyl ester derivative (Fura-2 AM). Cytosolic esterases split off the ester groups and leave the membrane-impermeant Fura-2 trapped in cytosol. This Fura-2 binds to Ca2+ present in cytosol and gives fluorescence (Figure 1). The procedure described here is similar to that of Cooper et al. (1985).
Pharmacological Control of Eosinophil Activation and Secretion
Published in Gerald J. Gleich, A. Barry Kay, Eosinophils in Allergy and Inflammation, 2019
Peter J. Barnes, Mark A. Giembycz
The fura-2 response to PAF is dependent on extracellular Ca2+, since it is reduced by bathing the cells in buffer without Ca2+ or in EDTA, which chelates extracellular Ca2+, but is not inhibited by the dihydropyridine calcium antagonist nimodipine, indicating that Ca2+ entry via voltage-dependent channels is not involved. The PAF-induced Ca2+ transient is, however, inhibited by nickel ions, which block Ca2+ entry via all channels, suggesting that occupation of PAF receptors opens receptor-operated channels, which allow the entry of Ca2+, resulting in cell activation. The concentration-response curve for the fura-2 response is similar to that for EPO release, suggesting that the rise in [Ca2+]i may be linked to degranulation, possibly via contractile myofilaments that are necessary for granule extrusion.
The neurosciences at the Max Planck Institute for Biophysical Chemistry in Göttingen
Published in Journal of the History of the Neurosciences, 2023
Neher and his colleagues were successful in further developing the patch clamp technique, and they were able to verify the fusion of vesicles with the cell membrane using capacitance measurement. Moreover, they filled the chromaffin cells with Ca2+ indicators (e.g., Fura-2) and this allowed them to optically measure the intracellular Ca2+ concentration.15Fura-2 is a fluorescent Ca2+ indicator. The principle using Fura-2 is based on the shift of its fluorescence excitation upon Ca2-binding. They used “caged Ca2+,” which is released through photolysis, to change the intracellular Ca2+ concentration quickly, and they were ultimately able to measure the release of catecholamines using a carbon microelectrode. By applying these four extremely complex and complementary methods (Figure 6), Neher and his colleagues were able to decipher the role of Ca2+ in vesicle release.
Resveratrol diminishes bisphenol A-induced oxidative stress through TRPM2 channel in the mouse kidney cortical collecting duct cells
Published in Journal of Receptors and Signal Transduction, 2020
Fura 2-AM calcium indicator, which specifically binds to the calcium inside the cell, was used to determine the intracellular calcium concentration [22]. Trypsin-treated cells were treated with 5 µM fura2-AM (Calbiochem, Darmstadt, Germany) in a 37 oC shaking water bath for 45 minutes after washing and centrifugation. The cell suspension (containing at least 1 million cell) treated with Fura 2-AM, a fluorescent marker based on ratiometric measurement, was placed in a 37 C transparent cuvette with magnetic stirrer in the Carry Eclipse spectrofluorometer (Varian Inc, Australia). Emission values of 505 nm were recorded at 340/380 nm excitations. Calcium amounts in cells were calculated according to Grynkiewicz method [28,29]. MpkCCDcl4 cells were stimulated with H2O2 (100 µM). TRPM2 channel activations were inhibited by ACA (25 µM).
Gintonin modulates platelet function and inhibits thrombus formation via impaired glycoprotein VI signaling
Published in Platelets, 2019
Muhammad Irfan, Dahye Jeong, Evelyn Saba, Hyuk-Woo Kwon, Jung-Hae Shin, Bo-Ra Jeon, Suk Kim, Sung-Dae Kim, Dong-Ha Lee, Seung-Yeol Nah, Man Hee Rhee
The [Ca2+]i concentration was assessed with Fura-2/AM as previously described (14). Briefly, platelets were pre-incubated with 5 µM Fura-2/AM for 1 h at 37°C. Following incubation, platelets were washed and treated with gintonin for 1 min in the presence of 1 mM CaCl2 at 37°C, followed by their stimulation with agonist for 2 min. Fluorescence was recorded using a spectrofluorometer (F-2500, Hitachi, Japan) and [Ca2+]i was calculated with Schaeffer and Blaustein’s method (15) using the following formula: [Ca2+]i in cytosol = 224 nM × (F−Fmin)/(Fmax−F), where 224 nM is the dissociation constant of the Fura-2-Ca2+ complex and Fmin and Fmax represent the fluorescence intensity levels at very low and very high Ca2+ concentrations, respectively.