**3. Results**

)

, thus reducing free

**2.2. Preparation of cells for Ca2+ imaging**

142 Calcium and Signal Transduction

and decreased a fraction of spontaneously oscillating cells.

**2.3. Ca2+ imaging and uncaging**

recorded from an individual cell.

uncaging. For Ca2+ or IP<sup>3</sup>

, 0.8 MgSO4

2 CaCl2

2 mM CaCl2

Before assaying with Ca2+ imaging, cells were maintained in a 12-socket plate for 12 h in the medium described above but without antibiotics. For isolation, cells cultured in a 1-ml socket were rinsed twice with the Versene solution (Sigma-Aldrich) that was then substituted for 200 μl HyQTase solution (HyClone) for 3–5 min. The enzymatic treatment was terminated by the addition of a 0.8 ml culture medium to a socket. Next, cells were resuspended, put into a tube, and centrifuged at 50 g for 45 s for moderate sedimentation. Isolated cells were collected by a plastic pipette and plated onto a photometric chamber of nearly 150 μl volume. The last was a disposable coverslip (Menzel-Glaser) with attached ellipsoidal resin wall. The chamber bottom was coated with Cell-Tak (BD Biosciences), enabling strong cell adhesion. Attached cells were then loaded with dyes for 20 min at room temperature (23–25°С) by adding Fluo-4 AM (4 μM) and Pluronic (0.02%; all from Molecular Probes) to a bath solution. Loaded cells were rinsed with the bath solution for several times and kept at 4°C for 1 h prior to recordings. Generally, incubation of MSCs at low temperature stabilized intracellular Ca2+

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>

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

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,

Ca2+ to nearly 260 nM at 23°С as calculated with the Maxchelator program (http://maxchelator.

in the bath was replaced with 0.5 mМ EGTA + 0.4 mМ CaCl<sup>2</sup>

uncaging, cells were loaded with 4 μM Fluo-4-AM (Invitrogen) and

, 10 glucose, 10 HEPES-NaOH, and pH 7.4 (≈270 Osm). When necessary,

In a typical experiment, nearly a hundred of MSCs loaded with Fluo-4 resided in a photometric camera, and their responsiveness to different ligands was assayed with Ca2+ imaging. Consistently with observations of others [3], functional heterogeneity was characteristic of a MSC population derived from each particular donor. Although a variety of GPCR agonists were found to stimulate Ca2+ signaling in MSCs, including ATP, ADP, noradrenaline or adrenaline, acetylcholine or its analog carbachol, GABA, glutamate, serotonin, and UTP, only a relatively small group of cells in a given MSC population was specifically responsive to a particular agonist (**Figure 1**). Overall, nearly 10<sup>3</sup> MSCs were sequentially stimulated by multiple agonists applied at different combinations, and a particular cell was either irresponsive to all stimuli or responded to one, rarely two, particular compound (**Figure 1A**–**C**). ATP-sensitive cells composed the most abundant subgroup of 9–15% (12% on average), depending on MSC preparation (**Figure 1B**). The percentage of cells responsive to other agonists was on average: ADP—7.1, adenosine—8.7, carbachol—3.4, GABA—5, glutamate—1.2, noradrenaline—6.7, serotonin—6.6, and UTP—6 (**Figure 1B**). The more or less accurate analysis of distribution of MSC responsivity was performed for nucleotides. In designated experiments, wherein cells were sequentially stimulated by ATP, ADP, and UTP, 125 purinergic MSCs were assayed overall, and only 13 cells (10%) were found to respond to all three agonists at the indicated concentrations (**Figure 1C**). Both ATP and ADP stimulated Ca2+ signaling in 40 cells (32%) that did not respond to UTP; 33 cells (26%) preferred the ATP-UTP pair. In addition, 20, 9, and 7 cells (16, 7, and 6%) responded exclusively to ATP, ADP, or UTP, respectively (**Figure 1C**).

Thus, the results presented above (**Figure 1**) clearly demonstrated that responsiveness to a given agonist varied from cell to cell. Note that GPCRs from most subfamilies, e.g. P2Y receptors, can couple to several signaling pathways, depending on cellular context [26–29]. Hence, in cells nonresponsive in terms of Ca2+ signaling to a particular agonist, appropriate GPCRs might be either not expressed or not coupled to Ca2+ mobilization.
