Xdi technology revolution multi point confocal system

HeLa Kyoto cell culture was imaged 24–48 h after transfection using a laser spinning-disk Andor XDi Technology Revolution multi-point confocal system (Andor, UK) equipped with an inverted Nikon Eclipse Ti microscope, a 75 W mercury-xenon lamp (Hamamatsu, Japan), a 60× oil immersion objective NA 1.4 (Nikon, Japan), a 16-bit QuantEM 512SC electron-multiplying CCD (Photometrics, USA), and a cage incubator (Okolab, Italy). Before imaging, the culture medium was changed to Dulbecco’s Phosphate Buffered Saline (DPBS) buffered with 20 mM HEPES, pH 7.4.
For time-lapse imaging experiments with varying Ca²⁺ concentration, 1 mM EDTA and 2.5 μM ionomycin were added to cells for imaging calcium indicators in the Ca²⁺-free state. After imaging calcium indicators in the apo-state, cells were washed with DPBS buffered with 20 mM HEPES, pH 7.4. Next, 2 mM CaCl₂ and 2.5 μM ionomycin were added to induce fluorescence signal for Ca²⁺-saturated calcium indicators.

HeLa Kyoto cell (kindly gifted by Belousov V.V from Moscow, IBC) cultures were imaged 24–48 h after transfection using a laser spinning-disk Andor XDi Technology Revolution multi-point confocal system (Andor, UK) equipped with an inverted Nikon Eclipse Ti microscope, a 75 W mercury-xenon lamp (Hamamatsu, Japan), a 60× oil immersion objective (Nikon, Japan), a 16-bit QuantEM 512SC electron-multiplying CCD (Photometrics, USA), and a cage incubator (Okolab, Italy). Before imaging, the culture medium was changed to Dulbecco’s Phosphate Buffered Saline (DPBS) buffered with 20 mM HEPES, pH 7.4.
For time-lapse imaging experiments with varying Ca²⁺ concentration, 1 mM EDTA and 5 μM ionomycin were added to cells for imaging calcium sensors in the Ca²⁺-free state. After imaging calcium indicators in the apo-state, cells were washed with DPBS buffered with 20 mM HEPES, pH 7.4. Next, 2 mM CaCl₂ and 5 μM ionomycin were added to induce fluorescence signal for Ca²⁺-saturated calcium indicators.