Orca flash 4.0 lt plus digital cmos camera

Single-cell Ca²⁺ imaging was used to measure cytoplasmic Ca²⁺ concentration according to our previous protocol [43 ]. Briefly, CF-7 cells were plated and cultured on coverslips to 80% confluency. Cells were then loaded with 2 μM Fura-2 AM in Hepes-buffered Hanks’ balanced salt solution (Hepes-HBSS) with 1% BSA for 30 min at room temperature. Fura-2 AM-loaded cells were mounted in an open bath imaging chamber (RC-21B; Warner Instruments) and visualized with a Nikon Eclipse Ti2 microscope (Nikon) using a 20X objective (Nikon CFI S Fluor Objective). Cells were alternately excited at 340 and 380 nm, and fluorescence emitted at 510 nm was captured by ORCA-Flash 4.0 LT PLUS Digital CMOS camera (Hamamatsu Photonics) every 5s controlled by Micro-manager [44 ]. Ratios of emitted fluorescence were determined pixel-to-pixel using ImageJ (NIH). During the Ca²⁺ imaging, ER or lysosomal Ca²⁺ stores were depleted by perfusing 1 μM TG (Thapsigargin) or GPN (glycyl-l-phenylalanine-β-naphthylamide, 400 μM) in Ca²⁺-free Hepes-HBSS, respectively and followed by re-addition of indicated concentration of SB202190. All the experiments were repeated 3 times, with at least 50 cells analyzed in each experiment as described by our previous report [43 ].