Fig. 6. Spontaneous Ca²⁺ oscillations were detected in HEK293 cells transfected with RyR3. (Aa) The time-course of [Ca²⁺]_i changes in HEK293 cell expressing RyR3 (black line) and a non-expressing cell (red line). Cells were sequentially treated with 1, 2, 5 and 10 mM caffeine and 1 µM ACh. (Ab) Ca²⁺ images in cells loaded with fura-2AM were obtained during resting conditions and in the presence of 1 mM caffeine or 1 µM ACh. The bottom-right panel in Ab indicates the immunofluorescence staining of RyR. It is noted that cells responding to 1 mM caffeine were stained with immunofluorescent RyR3 antibody. Bars in Ab, 20 µm. (B) The time courses of spontaneous [Ca²⁺]_i oscillations (a) were obtained from cells (1 and 2) as indicated in a series of confocal images (b). The Ca²⁺ images were obtained every 3 seconds, and cells were loaded with fluo-4 for 15 minutes. (C) Confocal images following fluo-4 loading (a) and staining with BODIPY FL-X ryanodine (b) in HEK293 cells transfected with RyR3. (a) Cells were loaded with fluo-4. [Ca²⁺]_i oscillations were then recorded in four indicated cells (1-4) among the 19 in this frame. (b) After recording [Ca²⁺]_i oscillation, cells were stained with BODIPY FL-X ryanodine for 5 minutes and washed. Among the 19 cells in the frame, 12 cells, including all four cells exhibiting [Ca²⁺]_i oscillations were well stained with BODIPY FL-X ryanodine. (c) The time courses of [Ca²⁺]_i oscillations of four cells in the frame.