Zepto b

The sensor Fluo-4 was loaded into Jurakt cells by incubating cells for 30 min in buffer (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 5 mM Mannitol, 10 mM HEPES, 2 mM CaCl₂, pH 7.3) containing 1 µM Fluo-4 AM (Life technologies, Carlsbad, CA, USA) on coated glass coverslips (Ø 25 mm). The latter were prepared by cleaning in a plasma furnace (Zepto-B, Diener electronic GmbH, Ebhausen, Germany) and coating with one layer of PBS/5% BSA in a spincoater (PIN150, SPS Europe Spincoating, Putten, Netherlands). After the initial layer had dried, it was further coated with a layer of poly-L-lysine (molecular weight 75–150 kDa). Coating was essential to prevent spontaneous Ca²⁺ oscillations, which usually occur when Jurkat cells are settling on glass coverslips. The dye was subsequently removed by washing cells with dye free buffer. After irradiation, the cells were then transferred for imaging on a Leica TCS SP5 II confocal microscope (Leica, Heidelberg, Germany) with a HCX PL APO CS 40.0 × 1.30 OIL oil immersion lens. The dye was excited with a 488 nm argon laser and the emission sampled at 505–550 nm.

Confocal laser scanning microscopy was performed on a Leica TCS SP or SP5 II system (Leica Microsystems, Mannheim, Germany) equipped with a 63× water (HCX PL APO 63× NA 1.2 W CORR) and 63 × 1.4 oil UV objective (HCX PL APO lambda blue). Coverslips were cleaned using acetone followed by plasma cleaning in a plasma furnace (Zepto-B) from Diener electronic (Ebhausen, Germany). The external buffer used for microscopy contained (140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 5 mM Mannitol, 10 mM HEPES, 2 mM CaCl₂, pH 7.4). Plasma membranes were imaged with CellMaskOrange™ (Thermo Fisher Scientific) at a concentration of 0.5 µg/ml. Nuclei were stained with Hoechst (200 µg/ml) diluted 1:50 in external microscopy buffer or PBS; cells were stained for 10 min at 37°C. Subsequently, cells were washed twice and resuspended in microscopy buffer or PBS.