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The use of the fluorescent dye Fura-2 to measure agonist- or OAG-induced TRPC activity: sample experimental protocols to measure the TRPC channel activity. The experimental details are described below. A complication in all these protocols is the native SOC activity. Although the native SOC activity can be inhibited by 1 μM Gd³⁺, Gd³⁺ can also inhibit at least in part the TRPC channel activity. Therefore, all the protocols below require controls to measure the native divalent ion fluxes and subtract them from the fluxes measured in cells expressing the desired TRPC channels. (a, left) An experiment aimed to measure the TRPC channel activity induced by stimulation of GPCR or by passive Ca²⁺ store depletion using the SERCA pump inhibitor thapsigargin and Ba²⁺ as a Ca²⁺ surrogate. In the beginning of the experiment, cells are placed in a Ca²⁺ free solution in order to separate Ca²⁺ release and influx events. Next, the agonist or thapsigargin is applied to deplete ER Ca²⁺ and activate the TRPC channels. After completion of Ca²⁺ release and return of Ca²⁺ to the basal level, a Ba²⁺-containing buffer is introduced; the amplitude of baseline deflection induced by Ba²⁺ application is a measure of the Ba²⁺ influx magnitude and, therefore, the activity of TRPC channels. Right: an experiment aimed to measure the TRPC activity stimulated by OAG using Ba²⁺ as a Ca²⁺ surrogate. In the beginning of the experiment, the cells are placed in a Ba²⁺-containing buffer in order to measure the magnitude of the basal Ba²⁺ influx. The small upward deflection of the baseline in this panel represents the basal Ba²⁺ influx. Next, OAG is added; the amplitude of baseline deflection induced by OAG application is a measure of the Ba²⁺ influx mediated by the OAG-activated TRPC channels. (Modified from Singh, B.B. et al., Molecular Cell 15, 635, 2004.) (b) An experiment aimed at measuring the TRPC channel activity induced by the ER Ca²⁺ store depletion using Mn²⁺ influx. Mn²⁺-induced quenching of Fura-2 is measured as a loss of Fura-2 fluorescence. Untreated cells were placed in a Mn²⁺-containing buffer in order to measure the magnitude of the basal Mn²⁺ influx (thin solid line). In parallel experiments, cells were treated with carbamylcholine, cyclopiazonic acid (CPA), or a combination of the two. The rate (slope) and the magnitude of Fura-2 quench induced by the Mn²⁺ influx are the measure of the Ca²⁺ channel activity in the PM. (Modified from Yao, J. et al., Journal of Biological Chemistry 279, 21511, 2004.) Sample setup. Buffers. The standard Fura-2 loading and cell bathing solution contains (in mM) 140 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES (pH 7.2). CaCl₂ is omitted in the Ca²⁺-free buffer; the Ba²⁺ buffer contains 2 mM Ba²⁺ instead of Ca²⁺. Buffers are applied by continuous perfusion (preferable) or by direct injection into the chamber accompanied by the immediate aspiration of the excess buffer. Each buffer application is equal to 5 to 10 chamber volumes in order to guarantee nearly complete replacement of the buffer in the chamber. Cell loading. The cells attached to cover slips are loaded with 1–5 μM Fura-2AM for 20–60 min at 37°C in a 5% CO₂ incubator and then incubated without Fura-2AM for 30 min to allow complete dye hydrolysis. The Fura-2 concentration and loading times differ depending on the cell type. The cells are used for experiments immediately after the loading. Fluorescent measurements. Experiments are performed in a dark room. The cover slip is placed in an experimental chamber and secured to the stage of an inverted microscope supplied with an objective capable of transmitting UV light, with an appropriate filter cube with a cutoff filter of <500 nm, source of excitation light, recording device, and software package. Fura-2 fluorescence is recorded at excitation wavelength of 340 and 380 nm (Ca²⁺ and Ba²⁺) or the isosbestic point of 360 nm (Mn²⁺). Light emitted at wavelength >500 nm is collected with a photomultiplier device or a CCD camera. Complete suitable recording systems are available from many vendors. Data interpretation. An increase in cytoplasmic Ca²⁺ or Ba²⁺ results in change in the Fura-2 fluorescence ratio recorded at 340/380 nm. Hence, fluorescence emitted by the sample that is being excited by a 340-nm light (excitation optima for Ca²⁺ bound Fura-2) increases, while fluorescence emitted by the sample that is being excited by a 380-nm light (excitation optima for Ca²⁺ free Fura-2) decreases. The ratio of fluorescence intensity induced by a 340-nm light over fluorescence intensity induced by a 380-nm light represents the fraction of Fura-2 bound to Ca²⁺ and, therefore, the cytoplasmic Ca²⁺ or Ba²⁺ concentrations. Consequently, plotting the ratio of fluorescence intensity against time yields the time profile of Ca²⁺ or Ba²⁺ changes in the cytoplasm. Ca²⁺ measurements obtained using this approach can be calibrated in terms of the absolute Ca²⁺ concentrations (see Chapter 15). In the experiment provided in the left panel of a, an agonist was applied while the cells were bathed in a Ca²⁺-free buffer. A spike in the fluorescence ratios caused by agonist application represents Ca²⁺ release from the ER Ca²⁺ store, as no Ca²⁺ influx from outside the cell is possible under these conditions. The stable increase in the fluorescence ratio induced by Ba²⁺ application illustrates the Ba²⁺ influx through active channels in the PM. The signal remains stable since the SERCA and PMCA cannot pump Ba²⁺. The magnitude of this influx can be compared between different samples either directly or normalized to the magnitude of Ca²⁺ release or Ba²⁺ influx in cells of which membranes were made permeable using the Ca²⁺ ionophore, ionomycin.