Andor xdi technology revolution multi point confocal system

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 cultures were imaged 24–48 h after the transient lipofectamine transfection before and immediately after 2.5 µM Ionomycin addition using a laser spinning-disk Andor XDi Technology Revolution multi-point confocal system (Andor Technology, Belfast, UK) as previously described [9 (link)].
For FRAP experiments, HeLa Kyoto cells were treated as described above and imaged using a Leica SP5 STED confocal microscope (Leica-Microsystems, Bensheim, Germany) and 70% of 488 nm laser power for bleaching (power at 100%—65 mW) during 1000 ms, with the capture settings: 100 ms per frame, 20 and 600 frames before and after bleaching, respectively, resolution—16 × 16 pixels, pixel size—0.38 µm.

HeLa Kyoto cells (kindly provided by Belousov V.V., Shemyakin‐Ovchinnikov Institute of Bioorganic Chemistry, Moscow) were imaged 24–48 h after the transient lipofectamine transfection before and after 2.5 μm Ionomycin addition using a laser spinning disk Andor XDi Technology Revolution multi‐point confocal system (Andor Technology, Belfast, UK) as previously described [5 (link)].
For fluorescence recovery after photobleaching (FRAP) experiments, HeLa Kyoto cells were transfected as described above and imaged using a Leica SP5 STED confocal microscope (Leica‐Microsystems, Bensheim, Germany) and 70% of 488 nm laser power for bleaching (power at 100%—65 mW) during 1000 ms, with the capture settings: 100 ms per frame, 20 and 600 frames before and after bleaching, respectively, resolution—16 × 16 pixels, pixel size—0.38 μm as described earlier [5 (link)].