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. Author manuscript; available in PMC: 2019 Apr 18.
Published in final edited form as: ACS Chem Neurosci. 2017 Dec 14;9(4):738–748. doi: 10.1021/acschemneuro.7b00369

Figure 4. Mitochondrial GSH concentration changes significantly in astrocytes but not in pyramidal cells in OHSCs during OGD-RP.

Figure 4

(a) The volumetric concentration of total GSH (mM) in OHSCs was sampled at five time points of 10/20/30 min basal/OGD/RP. Data are shown as mean ± SEM, n = 9 repeats for each case. Data in (c), (f), and (g) are acquired at the same time points as (a). (b–g) GSH in cells is labeled with the membrane-permeable, maleimide-based, fluorogenic Thiol probe IV (Ex: 405, Em: 450–480 nm). (b) OHSC exposed to 100 µM thiol probe IV for 8 min before imaging. (left) Nine pyramidal cells are outlined in white dash lines, while nine astrocytes are outlined in yellow solid lines. (right) The distribution of fluorescence intensity is shown in a false color pattern referencing to the color bar. The nine pyramidal cells and nine astrocytes are noted with (Δ) and (*), respectively. In (c, f, and g), the OGD period is indicated by a cyan box, and the RP period is right after it. (c) (left) The fluorescence intensity ratio of labelled GSH in astrocytes over pyramidal cells (FA/P) changes during OGD-RP. Data are shown as mean ± SEM (n = 38 cells for each case) with t test results (vs. the first data point (basal), p*** < 0.001, no label shown when p>0.05). (right) the GSH concentration in the two cell types. The GSH level is significantly higher in astrocytes than pyramidal cells. (d) Examples of a pyramidal cell in OHSC. Mitochondria (top) and whole cell (bottom) are visualized by Mito-GFP (Ex: 488, Em: 500–530 nm) and tdTomato (Ex: 561, Em: 580–600 nm), respectively. Column headings from left to right, FL: fluorescence image of fluorescent protein; Contour: the contour of the ratio of fluorescence from mitochondria/whole cell; Thiol Stain: overlay of the contour with the OHSC image (Before) and (After) GSH-staining. (e) Examples of an astrocyte are displayed in a similar way as in (d). (f) The fluorescence intensity ratios of labelled GSH in mitochondria over whole cell (FM/C) were record, and represented as mean ± SEM (n = 19 cells for each case). Each data point excluding the first one is compared with the first data point at the basal (**p<0.01, *p<0.05, no label shown when p>0.05) via t test. Red and black are used for pyramidal cells and astrocytes, respectively. (g) Volumetric concentrations of mitochondrial GSH are calculated from data in (a), (c), and (f) and shown as mean ± SEM (n = 9 repeats for each case). All images were acquired with 63× water-immersion lens with N.A. =0.9. Refractive Index of OHSC is around 1.36.