Lambda ls

Epifluorescence microscopy of live HeLa cells was performed 48 h after the transfection. HeLa cells were imaged using an Olympus IX81 inverted epifluorescence microscope equipped with a xenon-arc lamp Lambda LS (Sutter Instrument), 100× 1.4 NA oil immersion objective lens (UPlanSApo, Olympus), and Cy5.5 filter set (665/45 nm exciter and 725/50 nm emitter) (Chroma). SlideBook v.6 software (Intelligent Imaging Innovations) was used to operate the microscope.

For Ca²⁺ imaging studies, ARPE-19 cells at confluence were plated onto round coverslips covered with poly-l-lysine (10 μg/ml, Sigma Chemical) and incubated in the dark with 10 μM fluo 8-AM (Invitrogen) dissolved in the extracellular buffer containing: 150 mM NaCl, 20 mM HEPES, 0.1 mg/ml bovine serum albumin, 1.0 mM EGTA, adjusted to pH 7.4 with NaOH. Loaded cells were transferred into a perfusion chamber located on a microscope adapted to an epifluorescence system (Eclipse E600FN; Nikon Melville, NY). Cells were constantly perfused with oxygenated extracellular buffer at a rate of 15–17 ml/min and at 29–30 °C. Excitation of the fluorophore (at 488 nm) was performed with a light source controlled by a Lambda LS illuminator (Sutter Instruments, Novato, CA). Images were acquired with a cooled digital camera (Cool-SNAP-ES, Roper Scientific, Tucson, AZ) using the RS image software (Photometrics; Roper Scientific). The image field was 800 × 600 μm in size (Ramirez et al., 2012 (link)). Short movies (180 s total time, at 40 ms exposure at 4 Hz) were taken in control conditions and during the wash-in of the pharmacological treatments. To normalize the change in fluorescence of the treated cultures, their Δ fluorescence was corrected by subtracting that of one of the untreated conditions.