Cam xm10 cooled ccd camera

Patient-derived cultures of GBM cells, as well as U87MG cells, were grown in 96-well plates in media I and II at 37 ° C, 5% CO₂ atmosphere, 100% humidity. Before calcium imaging began, the growing medium was replaced with extracellular buffer (140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose, pH 7.4), and then each well was loaded with a cell-permeant 2,5 μM Fluo-4AM solution (ex/em = 494/506 nm; Thermo Fisher Scientific) for 40 min. The fluorescent dye solution was further removed, and cells were kept in an extracellular buffer for 1 h. Ca²⁺ dynamics were recorded using an Olympus IX71 epifluorescent microscope with an appropriate filter combination and a CAM-XM10 cooled CCD camera (Olympus, Tokyo, Japan). The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently. Videos were recorded and processed using CellA imaging software version 3.1 build 1274 (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Data analysis was performed using open-source ImageJ Fiji software (version 1.54f), where changes in fluorescent intensity per cell before and after the nAChR ligand exposure were calculated. The response of at least 5 cells was measured.

For measurements of intracellular calcium concentration [Ca²⁺]_i changes, transfected Neuro2a cells plated on glass coverslips were perfused at room temperature with the buffer containing 140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose; pH 7.4. Expression of Case12, a fluorescent genetically encoded sensor of calcium ions (ex/em = 491/516 nm), allowed direct monitoring of changes in [Ca²⁺]_i using an epifluorescence microscope with an appropriate filter combination and a CAM-XM10 cooled CCD camera (Olympus, Japan). Videos were made and processed using CellA Imaging Software (Olympus Soft Imaging Solutions GmbH, Germany), Image J, CellX, and OriginPro 7.5 software (OriginLab, MA, USA, for statistical analysis). The cells were exposed to acetylcholine iodide (Sigma, Germany), epibatidine (Tocris, UK), and α-cobratoxin purified from Naja kaouthia venom, and changes in Case12 fluorescence were recorded from each cell independently. To increase the registered changes, all ligand solutions contained the α7 nAChR positive allosteric modulator PNU120596 (10 μM, Tocris, UK). To allow recovery of the cells, the washing steps lasted 5–10 minutes. All recordings were made at room temperature.