Orca 03g digital ccd camera

Cells were cultivated on glass cover slips to 80 % confluency and then loaded with 1 µM FURA-2-AM (Sigma-Aldrich, St. Louis, MO, USA) in Calcium-free Tyrode buffer (0.1 mM EDTA, 138 mM NaCl, 1 mM MgCl₂, 5 mM KCl, 10 mM HEPES, 10 mM Glucose, pH 7.4) for 40 min at room temperature (RT) in the dark, after which they were washed and incubated in Calcium-free Tyrode buffer ± 10 µM Synta66 for 10 min at RT in the dark. During the recordings (Olympus IX71) using Live Acquisition v2.6 software (FEI, Planegg, Germany), cells were excited alternately using 340/26 and 380/11 nm filters (Semrock, Rochester, NY, USA) in an Oligochrome excitation system (FEI), and fluorescent images were captured using 510/84-nm emission filter (Semrock, Rochester, NY, USA) with an ORCA-03G digital CCD camera (Hamamatsu, Herrsching am Ammersee, Germany). The 340 nm/380 nm ratio was used as an index of cytosolic Ca²⁺ levels. In time-course experiments, fluorescent cells were recorded initially in a Calcium-free Tyrode solution followed by addition of 30 µM BHQ. Subsequently, fluorescent cells were recorded in a 2 mM Calcium Tyrode solution with BHQ. Analogous experiments were recorded in the presence of 10 µM Synta66.

Changes in intracellular Ca²⁺ ([Ca²⁺]_i) were monitored using red-shifted genetically-encoded Ca²⁺ sensor (R-GECO1.2). Briefly, HEK293 cells overexpressing R-GECO, WT–R-GECO or G652A–R-GECO were washed twice with experimental buffer (EB composition in mM: 140 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 glucose and 1 CaCl₂ (pH 7.4, adjusted with NaOH). The coverslip was then transferred to the imaging bath containing EB and 10 µM OptoBI-1 on an inverted microscope (Olympus IX71, Vienna, Austria) with 40 × 1.3 N.A. oil-immersion objective. Cells were excited to follow the R-GECO signal using 577/25 nm filters via TILL Oligochrome light source (TILL Photonics FEI Company, Graefelfing Germany) and fluorescent images were captured every second at 632 nm (using 632/60 nm emission filter, Chroma Technology, VT, USA) with an ORCA-03G digital CCD camera (Hamamatsu, Herrsching am Ammersee, Germany) using Live Acquisition 2.6 software (TILL Photonics FEI Company, Graefelfing Germany). The cis isomerization of OptoBI-1 compound was triggered by exposure to 10 s illumination period at 365 nm, and subsequent reversal of OptoBI-1 to trans conformation was achieved with 430 nm light exposure to 10 s period. The cycling between UV (365 nm; violet) and blue light (430 nm; blue) illuminations were employed one once or repeated three times. All experiments were performed at room temperature.

Changes in intracellular Ca²⁺ ([Ca²⁺]_i) were monitored using red-shifted genetically encoded Ca²⁺ sensor (R-GECO1.2). Briefly, RBL-2H3 cells overexpressing R-GECO with YFP-TRPC3, YFP-TRPC6 or TRPC7-CFP, were washed twice with experimental buffer (EB composition in mM: 140 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 glucose and 1 CaCl₂ (pH 7.4, adjusted with NaOH). Coverslips were mounted in a cell bath containing EB and 10 µM OptoBI-1 on an inverted microscope (Olympus IX71, Germany) with 40 × 1.3 N.A. oil-immersion objective. Cells were excited to follow the R-GECO signal using 577/25 nm filters via TILL Oligochrome light source (FEI, Germany) and fluorescent images were captured every second at 632 nm (using 632/60 nm emission filter, Chroma Technology, VT, USA) with an ORCA-03G digital CCD camera (Hamamatsu, Japan) using Live Acquisition 2.6 software (FEI, Germany). The cis isomerization of OptoBI-1 compound was triggered by exposure to 10 s illumination period at 365 nm, and subsequent reversal of OptoBI-1 to trans conformation was achieved with 430 nm light exposure to 10 s period. The cycling of UV (365 nm; violet) and blue light (430 nm; blue) illuminations were repeated three times. The interval between each illumination cycle was 60 s. All experiments were performed at room temperature. Detailed photocycling protocol is described in (26 (link)).