Cascade 512b ccd camera

LAD 2 cells were plated on poly-L-lysine (0.1 %)-coated coverslips, loaded with 1 μM Fura 2-AM and visualised on a Zeiss Axiovert microscope. Cells were perfused with imaging external solution containing (in mM) 142 NaCl, 5 NaHCO₃, 10 HEPES, 16 glucose, 2 KCl, 2 CaCl₂, 1 MgCl₂ and 0.1 % bovine serum albumin (BSA; pH 7.3, NaOH). Images were taken at 1-s intervals at 340 and 380 nm of light (15-ms exposure). The emitted light was passed through a 510–540-nm band pass filter before detection using a cascade 512B CCD camera (Roper Scientific, Photometrics UK). Data was collected and analysed using Metamorph® software (Meta Imaging), and further analysis and graphing were performed using Origin graphing software (OriginPro7.5, Origin corporation, USA). All fluorescence values are background subtracted and displayed as ratio changes. Data represents the mean ± standard error of mean (SEM) unless otherwise stated. Significance was assessed using a Student’s t test unless otherwise stated. In all figures, * = p < 0.05, ** = p < 0.01. Responding cells were counted as those where calcium levels in response to drug rose by more than 5 standard deviations over the baseline in a specified period of time. All concentrations of agonists used were based on previous experiments demonstrating the functional presence of P2X receptors in human mast cells [25 (link)].