Glass bottom μ dishes

Cortical neurons were obtained from the cortical lobes of E18 Sprague-Dawley rat embryos according to previously described procedures [19 (link)]. Neurons were resuspended in B27 Neurobasal medium plus 10% FBS and then seeded onto poly-L-ornithine-coated 48-well plates or glass coverslips (12 mm in diameter) at 1.5 × 10⁵ cells per well. For confocal single-cell imaging experiments, cells were plated onto glass-bottom μ-dishes (Ibidi GmbH, Germany). The medium was replaced by serum-free, B27-supplemented Neurobasal medium 24 hours later. The cultures were essentially free of astrocytes and microglia and were maintained at 37°C and 5% CO₂ as was previously described [25 (link), 26 (link)].
For transfection of cells, 4 × 10⁶ rat neurons were transfected in suspension before plating with 3 μg of cDNA using the Rat Neuron Nucleofector kit (Lonza, Switzerland) according to the manufacturer instructions and plated and maintained as described above. Cultures were used at 8–9 days in vitro.

The intracellular calcium ion concentration, [Ca²⁺]i, was measured using a conventional Fura-2 technique as previously described.^{11 (link)} After 48 h of transfection with pcDNA, wtSigR1 or mSigR1, NSC-34 and MCF-7 cells cultured in glass bottom μ-dishes (ibidi), were loaded with the membrane-permeable AM-form of Fura-2 (1.5 ng/μl; Invitrogen) in the presence of pluronic acid (25 %) for 30 min at 37 °C. Emitted fluorescence at 530 nm (detected using a PCO (Sensicam: pco.imaging), Kelheim, Germany) in response to alternate excitation at 340 nm and 380 nm (using the Polychrome V monochromator; TILL Photonics, Gräfelfing, Germany) was used to measure intracellular Ca²⁺ concentrations. Data were shown as emission ratios in response to 340 nm /380 nm excitation. Whole-cell calcium measurements of NSC-34 and MCF-7 cells transfected with pcDNA, wtSigR1 or mSigR1 were obtained at room temperature (23–25 °C). During the imaging procedure, cells were kept in a bathing solution containing 100 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 MES, 5.5 glucose and pH was adjusted to 7.4.