FIG. 3.

Similar reduced thiol concentration in mitochondria or whole cell between CA1 and CA3 pyramidal cells. (A) Fluorescence imaging begins before exposure of cultures to the fluorogenic reagent thiol probe IV. The reaction occurs in seconds. Nonfluorescent thiols turn fluorescent after exposure to 100 μM thiol probe IV (top). The increasing fluorescence rising to a steady value is indicative of the reaction occurring during the rise and then ceasing when the thiols are all reacted. Representative images from CA1 (bottom) are displayed: (left) BF image, (right) fluorescence image (ex 405 nm, em 440–480 nm). Imaging protocol is optimized as shown in Supplementary Figure S2. (B) Threshold was set to the fluorescence image in (A) to define the areas of pyramidal cells (gray), above threshold (green), and below threshold (blue). CA1 and CA3 pyramidal cells reach a constant fluorescence after about 8 min, indicating that the reaction is complete. (C) A mask of mitochondria is created from a pyramidal cell expressing MitoGFP in CA1 (left) and applied to count the thiol fluorescence in mitochondria (right). (D) The CA1/CA3 ratio of reduced thiols in mitochondria and, separately, whole cell is ∼1.0 (n = 17 OHSCs). BF, bright-field.