Metafluor computer software

Fluorescence measurements were performed using an inverted phase-contrast microscope (Axiovert 100; Zeiss, Oberkochen, Germany). Fluorescence was evoked by a filter wheel (Visitron Systems, Puchheim, Germany) mediated alternative excitation at 340/26 or 387/11 nm (AHF, Analysentechnik, Tübingen, Germany). Excitation and emission light was deflected by a dichroic mirror (409/LP nm beamsplitter, AHF) into the objective (Fluar x40/1.30 oil; Zeiss) and transmitted to the camera (Visitron Systems), respectively. Emitted fluorescence intensity was recorded at 587/35 nm (AHF). Excitation was controlled and data acquired by Metafluor computer software (Universal Imaging, Downingtown, PA, USA). The 340/380 nm fluorescence ratio was used as a measure of cytosolic free Ca²⁺ concentration (_free[Ca²⁺]_i). The cells were irradiated (0 or 5 Gy) and loaded with fura-2/AM (2 μM for 30 min at 37°C; Molecular Probes, Goettingen, Germany) in supplemented RPMI medium. _free[Ca²⁺]_i was determined 1.5–5 h after IR at 37°C during superfusion with NaCl ringer (see above), upon Ca²⁺ depletion with Ca²⁺-free NaCl ringer solution (in mM: 125 NaCl, 32 HEPES, 5 KCl, 5 d-glucose, 1 MgCl₂, and 0.5 EGTA, titrated with NaOH to pH 7.4), and during Ca²⁺ readdition in NaCl ringer solution additionally containing ACA (0 or 20 μM).

Fluorescence measurements were performed at 37°C using an inverted phase-contrast microscope (Axiovert 100; Zeiss, Oberkochen, Germany). Fluorescence was evoked by a filter wheel (Visitron Systems, Puchheim, Germany)-mediated alternative excitation at 340/26 or 387/11 nm (AHF, Analysentechnik, Tübingen, Germany). Excitation and emission light was deflected by a dichroic mirror (409/LP nm beam splitter, AHF) into the objective (Fluar x40/1.30 oil; Zeiss) and transmitted to the camera (Visitron Systems), respectively. Emitted fluorescence intensity was recorded at 587/35 nm (AHF). Excitation was controlled and data acquired by Metafluor computer software (Universal Imaging, Downingtown, PA, USA). The 340/380-nm fluorescence ratio was used as a measure of cytosolic free Ca²⁺ concentration (_free[Ca²⁺]_i). TRPM8- , MSI1- or nt siRNA-transfected U251 cells (24 h (MSI1) and 48 h (TRPM8) after transfection) were incubated with fura-2/AM (2 μM for 30 min at 37°C; Molecular Probes, Goettingen, Germany) in DMEM/10% FCS medium, respectively. Steady state _free[Ca²⁺]_i was recorded in Ca²⁺-containing NaCl solution (see above) before and during superfusion of icilin (10 μM).

Fluorescence measurements were performed by the use of an inverted phase-contrast microscope (Axiovert 100; Zeiss, Oberkochen, Germany). Fluorescence was evoked by a filter wheel (Visitron Systems, Puchheim, Germany)-mediated alternative excitation at 340/26 or 387/11 nm (AHF, Analysentechnik, Tübingen, Germany). Excitation and emission light were deflected by a dichroic mirror (409/LP nm beamsplitter, AHF) into the objective (Fluar x40/1.30 oil; Zeiss) and transmitted to the camera (Visitron Systems), respectively. Emitted fluorescence intensity was recorded at 587/35 nm (AHF). Excitation was controlled and data acquired by Metafluor computer software (Universal Imaging, Downingtown, PA, USA). The 340/380-nm fluorescence ratio was used as a measure of cytosolic free Ca²⁺ concentration (c[Ca²⁺]_free). K562 cells were irradiated (0 or 5 Gy) and loaded with fura-2/AM (2 µM for 30 min at 37°C; Molecular Probes, Göttingen, Germany) in supplemented RPMI medium. Steady state c[Ca²⁺]_free was recorded in irradiated (0 or 5 Gy) K562 cells (1–4 h post-irradiation) in the continuous presence of external Ca²⁺ during superfusion with Ca²⁺-containing NaCl solution (see above) before, during, and after administration of E4013 (1µM) or TEA (3 mM).