Evolve 512 delta

Imaging experiments were performed with a Zeiss Cell Observer Z1 equipped with a 40× oil Fluar (N.A. 1.3) objective, multi-filter system, fast acquisition EMCCD camera (Evolve^®512 Delta) and LED system (Colibri, Zeiss) at 37°C. Data were analyzed with AxioVision software (Zeiss, Oberkochen, Germany). HeLa cells were cultivated in DMEM medium (Gibco^TM, Cat: 11965084) supplemented with 10% FCS at 37°C in 5% CO₂. About 2.5*10⁵ HeLa cells were seeded on 25 mm round glass coverslips for 12 h before transfection of DNA constructs. Mover-IRES-mKate, Mover F206R-IRES-mKate or Mover4mut-IRES-mKate and NFAT1-GFP plasmid were co-transfected using Fugene^® HD (Promega GmbH, Mannheim, Germany) and Opti-MEM^TM (Gibco^TM, Cat: 31985070) along with 1 μg of plasmid DNA according to the manufacturer’s instructions, 48 h before the imaging experiment. During the experiment, HeLa cells were perfused with Ringer’s buffer containing 1 mM Ca²⁺ and activated by 10 μM histamine (Sigma, H7125), as indicated. NFAT1-GFP fluorescence was recorded using LED diode (excitation 505 nm) and emission filter (525 ± 25 nm). The increase of fluorescence intensities in the nucleus was marked with region of interest (ROI) and analyzed by normalizing the background-corrected fluorescence intensity. Mann–Whitney test was used for testing the significance of the results.