Uapon340 40 oil immersion objective

Cytosolic Ca²⁺ measurement without UV light was performed with an Olympus IX73 inverted microscope. This microscope was equipped with an UApoN340 40× oil immersion objective (Olympus, Tokyo, Japan) and a CCD Retiga R1 camera (Q-imaging, Vancouver, BC, Canada). LedHUB^® (Omnicron, Rodgau, Germany) equipped with 340, 385, 455, 470, and 550 nm LEDs. A GFP (GFP-3035D, Semrock, Henrietta, NY, USA) filter set was used for illumination of Fluo-4. Visiview 4.2.01 (Visitron, Puchheim, Germany) was used for the data acquisition. Alternatively, an AnglerFish F-G/O (Next Generation Fluorescence Imaging/NGFI (www.ngfi.eu), Graz, Austria) was used for data acquisition. Subsequent data analysis was performed in ImageJ (NIH, Bethesda, MD, USA) and Excel (Microsoft, Redmond, WA, USA). Both microscopes were equipped with an automatic perfusion system PS-9D (NGFI).
To avoid usage of UV light, cells were loaded with 3.3 µM Fluo-4 in EB for 30 min. On the microscope, cells were perfused with 2 mM Ca²⁺ buffer for 24 min and 2-min 30 mM K⁺ buffer to get maximum uptake through the L-type Ca²⁺ channels. The depolarization induced maximum Ca²⁺ uptake via L-type Ca²⁺ channel is used for the normalization of Fura-2. The number of cytosolic Ca²⁺ spikes was calculated from min 2 to min 24.