Cam xm10 charge coupled device ccd

Cells transferred to poly-L-lysine covered glass slips were incubated for 2 h at 37 °C. The cells adhered to glass were further incubated with Fluo-4AM given at 2 mM and organic anion transporter inhibitor probenecid at 1.25 mM for 1 h at room temperature. After incubation, cells were washed out with extracellular solution (140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose; pH 7.4). The last wash-out was supplied with 10 µM PNU 120,596 (an α7 nAChR positive allosteric modulator), and, then, Fluo-4 was excited at 485 nm and fluorescence registered at 535 nm. The measurements were performed on Olympus (Japan) epifluorescence microscope with a CAM-XM10 charge-coupled device (CCD). CellA Imaging Software (Olympus Soft Imaging Solutions GmbH, Germany) was used to record video further analyzed with ImageJ. PNU 282,987 (1 µM) was added directly to the cells preparations. The α7 nAChR inhibition was achieved by 10 µM α-bungarotoxin application 5 min before adding the agonist PNU 282,987. The calcium rise was measured relative to the fluorescence base level of each cell.

MDMs were plated on poly-L-lysine slides in FluoroBrite DMEM Media and stained with Alexa Fluor-555 α-bungarotoxin (100 nM) for 30 min at 37°C (all from Thermo Fisher Scientific, Waltham, MA, USA). Slides were washed, fixed in buffered 4% paraformaldehyde, coated with a carbonate-buffered glycerin, and examined with an Olympus (Japan) epifluorescence microscope with a CAM-XM10 charge-coupled device (CCD) equipped with appropriate filter sets. Images were analyzed with the ImageJ software (NIH, USA).