Fluar 20 0.75 objective

For cell fluorescence measurements, GSC lines were grown on glass coverslips mounted in a cell chamber and perfused with bath solution at room temperature. Fluorescence was measured continuously on an inverted microscope (IX71, Olympus, Chromaphor) using a Fluar 20 ×/0.75 objective (Olympus) and Till Vision real-time imaging software (Till Photonics). Cells were loaded for 15 min at 37 °C with 2 μM Fura-2-AM (Molecular Probes) in bath solution. Fura-2 was excited at 340/380 nm, and the emission was recorded between 470 and 550 nm using a sensicam CCD camera (PCO imaging). Acquisition and data analysis were done using Till Vision software. Ionomycin, capsaicin, menthol and ATP were purchased from Sigma-Aldrich.

For cell fluorescence measurements, LUHMES cells grown on glass coverslips were mounted in a cell chamber and perfused with bath solution at room temperature. Fluorescence was measured continuously on an inverted microscope (IX71, Olympus, Chromaphor) using a Fluar 20×/0.75 objective (Olympus) and Till Vision real-time imaging software (Till Photonics). Cells were loaded for 15 min at 37°C with 2 μM Fura-2-AM (Molecular Probes) in bath solution. Fura-2 was excited at 340/380 nm, and the emission was recorded between 470 and 550 nm using a sensicam CCD camera (PCO imaging). Acquisition and data analysis were done using Till Vision software. Amiloride and nimodipine were purchased from Sigma–Aldrich.

To determine intracellular Ca²⁺-concentrations, DAOY cells were seeded on cover slips, mounted in a cell chamber and perfused as described in the section “Whole-cell patch clamp”. Fluorescence was measured every 2 s on an inverted microscope (IX71, Olympus, Chromaphor) using a Fluar 20 × /0.75 objective (Olympus) and Till Vision real-time imaging software (Till Photonics). Cells were loaded for 30 min at 37 °C with 2 μM Fura-2-AM (Molecular Probes) in the bath solution. Fura-2 was excited at 340/380 nm and the emission was recorded between 470 and 550 nm using a sensicam CCD camera (PCO imaging). Acquisition and data analysis were done using the Till Vision software and Excel.
The bath solutions used for the capsaicin, menthol, ATP and ionomycin stimulations consisted of the conditioning bath solution described under “Whole-cell patch clamp” containing the following chemicals: 100 μM capsaicin (Sigma Aldrich), 200 μM menthol (Sigma Aldrich), 100 μM ATP (Sigma Aldrich), or 1 μM ionomycin (Santa Cruz Biotechnology, Dallas, USA), respectively. The 30 mM K⁺ solution contained the following compounds (in mM): NaCl 85, KCl 30, D-glucose 5, HEPES 5, glucose 5.5, MgCl₂ 1, sodium gluconate 25, calcium gluconate 3. pH was adjusted to 7.4 with NAOH and HCl. The pH 6.0 solution consisted of the acidic bath solution described in “Whole-cell patch clamp”.