**2.3. Ca2+ imaging and uncaging**

Experiments were carried out using an inverted fluorescent microscope Axiovert 135 equipped with an objective Plan NeoFluar 20x/0.75 (Zeiss) and a digital EMCCD camera LucaR (Andor Technology). Apart from a transparent light illuminator, the microscope was equipped with a handmade system for epi-illumination via an objective. The epi-illumination was performed using a bifurcational glass fiber. One channel was used for Fluo-4 excitation and transmitted irradiation of a computer-controllable light-emitting diode (LED) LZ1-00B700H (LED Engin). LED emission was filtered with an optical filter ET480/20x (Chroma Technology). Fluo-4 emission was collected at 535 ± 25 nm by using an emission filter ET535/50 m (Chroma Technology). Serial fluorescent images were usually captured every second and analyzed using Imaging Workbench 6 software (INDEC). Within the 1-s acquisition period, the 480 nm LED was switched on for only 200 ms, during which cell fluorescence was collected. This protocol allowed for minimizing photobleaching of Fluo-4 at a sufficiently high signal-to-noise ratio achievable by adjusting LED emission. This enabled us to reliably assay cell responsiveness to different compounds applied serially for up to 60 min. Deviations of cytosolic Ca2+ from the resting level were quantified by a relative change in the intensity of Fluo-4 fluorescence (ΔF/F<sup>0</sup> ) recorded from an individual cell.

Another channel was connected to a pulsed solid laser TECH-351 Advanced (680 mW) (Laser-Export, Moscow). This unit operated in a two harmonic mode and generated not only 351 nM UV light used for Ca2+ uncaging but also visible light at 527 nm. The last could penetrate into an emission channel through nonideal optical filters and elicit optical artifacts during uncaging. For Ca2+ or IP<sup>3</sup> uncaging, cells were loaded with 4 μM Fluo-4-AM (Invitrogen) and 4 μM NP-EGTA-AM (Invitrogen) or 4 μM caged-Ins(145)P3/PM (SiChem) + 0.02% Pluronic (Invitrogen) for 30 min at 23°С. The basic bath solution contained (mM): 110 NaCl, 5.5 KCl, 2 CaCl2 , 0.8 MgSO4 , 10 glucose, 10 HEPES-NaOH, and pH 7.4 (≈270 Osm). When necessary, 2 mM CaCl2 in the bath was replaced with 0.5 mМ EGTA + 0.4 mМ CaCl<sup>2</sup> , thus reducing free Ca2+ to nearly 260 nM at 23°С as calculated with the Maxchelator program (http://maxchelator.

stanford.edu). In this low Ca2+ bath solution, the glucose concentration was increased to 13 mM to keep osmolarity. All chemicals used in experiments described below were applied by the complete replacement of the bath solution in a 150-μl photometric chamber for nearly 2 s using a perfusion system driven by gravity. The used salts and buffers were from Sigma-Aldrich, and agonists and inhibitors were from Tocris.
