75 w mercury xenon lamp

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.

Mammalian live cell imaging was performed as described earlier [28 (link)]. Briefly, transient transfection of the HeLa Kyoto cells was performed in a 24-well format using lipofectamine reagent according to the manufacturer’s protocol. Cells were cultured using DMEM medium supplemented with 10% FBS, glutamine, 50 U/mL penicillin, and 50 U/mL streptomycin, at 37 °C and 5% CO₂. HeLa cell cultures were imaged 24–72 h after the transient transfection using a laser spinning-disk Andor XDi Technology Revolution multi-point confocal system (Andor Technology, Belfast, UK) equipped with an inverted Nikon Eclipse Ti-E/B microscope (Nikon Instruments, Tokyo, Japan), a 75 W mercury–xenon lamp (Hamamatsu, Hamamatsu, Japan), a 60× oil immersion objective NA 1.4 (Nikon, Tokyo, Japan), a 16-bit Neo sCMOS camera (Andor Technology, Belfast, UK), a laser module Revolution 600 (Andor Technology, Belfast, UK), and a spinning-disk module Yokogawa CSU-W1 (Andor Technology, Belfast, UK). The blue, green, and red fluorescence were acquired using the 405, 488, and 561 nm lasers, a confocal dichroic mirror 405/488/561/640, and filter wheel emission filters 447/60, 525/50, and 617/73, respectively. During imaging, the cells were incubated at 37 °C and 5% CO₂ using a cage incubator (Okolab, Naples, Italy).

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.