PMC:4925210 / 29045-31804
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4925210","sourcedb":"PMC","sourceid":"4925210","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4925210","text":"Ratiometric single cell Ca2+ imaging\nCHO-K1 cells were plated onto glass coverslips and placed in 35-mm dishes where they were transfected with full length mouse PKD1, TRPP2, and yellow fluorescent protein (YFP) cDNAs using Lipofectamin2000 (Invitrogen). Twenty four hours after transfection cells were loaded with 2 μM Fura-2/AM in extracellular solution (ECS) containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, and 15 mM Hepes, pH 7.4, ([Ca2+]o: 1.8mM) in the presence of 0.05% Pluronic F-127 for 45 min at room temperature. Cells were washed twice in ECS and incubated for 15 min in 37° C before intracellular imaging. Coverslips were mounted onto the stage of an Axiovert200 inverted microscope and cells were perfused with ECS or 500 ng/ml WNT9B in ECS for 30 min. Ionomycin (2 μM) was added towards the end of the experiment (time point 35 min) to induce maximum Ca2+ entry. To determine the effect of WNT9B on transfected cells in nominally Ca2+-free extracellular solution, cells were perfused with ECS for 5 min and then ECS was switched to a Ca2+-free extracellular solution (same as ECS but without CaCl2 plus 10μM EGTA) with or without WNT9B (500 ng/ml) for 30 min. Ca2+ (1.8 mM) was added back at time point 35 min. Singly transfected cells were identified using a YFP filter set and imaged using a Fura-2 filter set with a CCD camera (CoolSnap HQ, Photomertrics) driven by the Metafluor software (Universal Imaging). Fluorescence ratios of 340/380 were taken every 1 s using 200 ms exposure time. Changes in intracellular Ca2+ concentration (Δ[Ca2+]i) was expressed as 340/380 ratio. Traces represent average fluorescence ratio 340/380 over time from 50-65 cells pooled from 3-5 independent experiments.\nPkd2+/+ and Pkd2−/− MEFs were plated onto glass coverslips. Twenty four hours after seeding, cells were loaded with 2 μM Fura-2/AM in Ca2+-free extracellular solution (same as ECS but without CaCl2 plus 10μM EGTA) in the presence of 0.05% Pluronic F-127 for 45 min at room temperature. Cells were washed twice in ECS-Ca2+ free and incubated for 15 min in 37° C before intracellular imaging. Coverslips were mounted onto the stage of an Axiovert200 inverted microscope and cells were treated with 500 ng/ml WNT9B followed by 100 μm ATP at the end of experiment. Cells (47 for Pkd2+/+, 37 for Pkd2−/−) were imaged using a Fura-2 filter set with a scientific CMOS camera (Pco.Edge Scientific) controlled by EasyRatioPro software (PTI). Fluorescence ratios of 340/380 were taken every 1 s using 200 ms exposure time. Changes in intracellular Ca2+ concentration (Δ[Ca2+]i) was expressed as 340/380 ratio. Traces represent average fluorescence ratio 340/380 over time from 37-47 cells pooled from 3 independent experiments.","divisions":[{"label":"Title","span":{"begin":0,"end":36}}],"tracks":[]}