Cary eclipse fluorescence spectophotometer

UV measurements were made in a Jasco V-630 spectrophotometer coupled to a Jasco ETC-717 temperature controller, using a standard Hellma semi-micro cuvette (108.002-QS) with a light path of 10 mm. Measurements were made at 20°C. Luminescence experiments were made with a Varian Cary Eclipse Fluorescence Spectophotometer coupled to a Cary Single Cell peltier accessory (Agilent Technologies) temperature controller. All measurements were made with a Hellma semi-micro cuvette (108F-QS) at 20°C. Circular dichroism measurements were made with a Jasco J-715 coupled to a Neslab RTE-111 termostated water bath, using a Hellma 100-QS cuvette (2 mm light pass).

Changes in plasma membrane permeability on exposure to BzATP (200 μM) (Sigma-Aldrich) were studied by ethidium bromide uptake. One hundred thousand cells were kept at 37° C in a thermostat-controlled and magnetically stirred cuvette of a Cary Eclipse Fluorescence Spectophotometer (Agilent Technologies) in the presence of 20 μM ethidium bromide (Sigma-Aldrich). Fluorescence changes were acquired at 360 nm and 580 nm excitation and emission wavelengths, respectively.
For A172, M059J, T98G, U87MG, 8 × 10⁴ cells were seeded in 96-well plates for 24 h, washed with PBS, incubated for 10 min in PBS supplemented with ethidium bromide (5 μg/mL) and 200 μM BzATP. A positive control of ethidium bromide incorporation was performed treating cultures with permeable buffer solution during ethidium bromide exposure (3.5 mM trisodium citrate, 0.5 mM Tris buffer and 0.1 % nonidet). ethidium bromide fluorescence was observed using a inverted fluorescence microscope (Nikon Eclipse TE300; Melville, NY, USA). The exposure time used to acquire images from treated samples was the same used for acquisition of images from permeabilized cells (maximal ethidium bromide uptake).

The cytosolic Ca²⁺ concentration was measured using the fluorescent Ca²⁺ indicator Fura-2-acetoxymethyl ester (Fura-2/AM) (Thermo Fisher Scientific) [55 (link)]. Cells were incubated at 37° C for 20 minutes in sodium solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM NaH₂PO₄, 20 mM HEPES, 5.5 mM glucose, 1 mM CaCl₂, pH 7.4) supplemented with 4.0 μM Fura-2/AM and 250 μM sulfinpyrazone (Sigma-Aldrich). In experiments performed in the absence of external Ca²⁺, CaCl₂ was omitted and 1.0 mM EGTA was added._. Cells were kept at 37° C in a thermostat-controlled and magnetically-stirred cuvette of a Cary Eclipse Fluorescence Spectophotometer (Agilent Technologies, Milan, Italy) and [Ca²⁺]_i was determined at the 340/380nm excitation ratio and at 505 nm emission wavelengths. ATP (Sigma-Aldrich), BzATP (Sigma-Aldrich) or A740003 (Tocris Bioscience, Bristol, UK) were added when indicated.