ethd‐1(ethidium homodimer‐1, 2 μ m )  (Thermo Fisher)

Journal: The Journal of Neuroscience

Article Title: Dissipation of Potassium and Proton Gradients Inhibits Mitochondrial Hyperpolarization and Cytochrome c Release during Neural Apoptosis

doi: 10.1523/JNEUROSCI.21-13-04551.2001

Figure Lengend Snippet: Overexpression of Bcl-xL inhibits staurosporine-induced mitochondrial hyperpolarization and cytochrome c release. a–d, D283 cells were exposed to vehicle or STS. After 30 min exposure to STS, cytochrome c immunofluorescence remained mitochondrial (d), whereas CMXRos uptake increased significantly (b). Scale bar, 10 μm. Experiments were performed three times with comparable results.e–l, D283 medulloblastoma cells were stably transfected with pSFFV-Neo-Bcl-xL (D283/Bcl-xL) or empty plasmid pSFFV-Neo (D283/Neo). Overexpres- sion of Bcl-xL was confirmed by immunoblotting using an anti-Bcl-x antibody (f, inset).e, Quantification of CMXRos fluorescence in D283/Neo cultures confirmed an early increase in CMXRos uptake after the exposure to STS. In contrast, the increase of CMXRos uptake was inhibited in D283/Bcl-xL cells. Data are means ± SEM fromn = 8 cultures; *p < 0.05 with respect to control. Experiment was performed in triplicate and yielded comparable results. f, Quantification of TMRE uptake in D283/Neo cultures confirmed the early increase of fluorescence obtained with CMXRos. The increase of TMRE uptake was inhibited in D283/Bcl-xL cells. Data are means ± SEM from n = 8 cultures; *p < 0.05 with respect to control. Experiment was repeated twice with comparable results.g–j, Cytochrome c distribution was visualized by immunofluorescence analysis after 3 hr exposure to STS or vehicle. Note that the STS-induced cytochrome c release was significantly reduced in D283/Bcl-xL cells (j) compared with D283/SFFV cells (h). Scale bar, 10 μm (g). k, Control cells and Bcl-xL-overexpressing cells were treated with vehicle (Con) or STS for 4 and 6 hr. Caspase-3-like protease activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Activities are presented as increase in AMC fluorescence (in arbitrary fluorescence units) over 60 min per microgram of protein. Data are means ± SEM from n = 8 cultures. The experiment was repeated three times with similar results. Different from controls, *p < 0.05.l, Quantification of cell death after exposure to STS. D283 cells were incubated with STS or vehicle for up to 24 hr. Cells were stained simultaneously with 1 μm calcein AM and 2 μm ethidium homodimer (EthD-1). Live (green) and dead (red) cells were counted. n = 4 cultures (430–3000 cells) per time point. Different from controls, *p < 0.05.

Article Snippet: Cells were simultaneously stained with 1 μ m calcein AM and 2 μ m ethidium homodimer (EthD-1) in serum-free medium using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes).

Techniques: Over Expression, Immunofluorescence, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Fluorescence, Activity Assay, Incubation, Staining

Journal: The Journal of Neuroscience

Article Title: Dissipation of Potassium and Proton Gradients Inhibits Mitochondrial Hyperpolarization and Cytochrome c Release during Neural Apoptosis

doi: 10.1523/JNEUROSCI.21-13-04551.2001

Figure Lengend Snippet: Overexpression of Bcl-xL does not inhibit valinomycin-induced mitochondrial depolarization and cytochrome c release. a–d, Representative images of vehicle-treated (a, c) and Val-treated (b, d) D283 medulloblastoma cells fixed after 15 min of exposure. a,b, Changes of mitochondrial membrane potential were determined by CMXRos uptake. Note that the mitochondrial staining pattern was lost after treatment with Val (b).c, d, Distribution of cytochrome c was determined by immunofluorescence analysis. Cytochrome c immunofluorescence remained intact after exposure to Val (d). Scale bar, 10 μm. e, Quantification of cell death after exposure to Val. D283/Neo and D283/Bcl-xL cells were exposed to vehicle or Val for 12 and 24 hr and stained simultaneously with 1 μm calcein AM and 2 μm ethidium homodimer. The percentage of dead cells was determined. n = 4 cultures (600–3000 cells) per time point. Different from controls, *p < 0.05.n.s., Not statistically significant.f–m, Cells were treated with Val or vehicle for 6 hr. Cytochrome c distribution was visualized by immunofluorescence analysis. Val-induced cytochrome c release was not inhibited in Bcl-xL-overexpressing cells (l, arrowheads) compared with control cells (h). Mitochondria appeared swollen in control and Bcl-xL-overexpressing cells (h, l; arrows). Overexpression of Bcl-xL could also not inhibit loss of mitochondrial membrane potential (i, m). Scale bar (in f): f–m, 10 μm.n–p, High magnification of mitochondria and mitochondria-rich regions in vehicle- and Val-treated cultures (6 hr) stained with the cytochrome c antibody. Overexpression of Bcl-xL does not inhibit large-scale mitochondrial swelling induced by Val. Mitochondria of vehicle-treated D283/Neo and D283/Bcl-xL cells were indistinguishable, therefore only mitochondria of a D283/Bcl-xL cell are shown. Images were deconvoluted using No Neighbor Deblurring software, which applies the algorithm of Monck et al. (1992) to reduce image background haze attributable to light originating from unsharp areas of the specimen. Scale bar (in n): n–p; 5 μm. N, Nucleus. All experiments were repeated twice with comparable results. q–r, Images of mitochondria in vehicle- and Val-treated (6 hr) rat primary astrocytes stained with the anti-cytochrome c antibody. Images were deconvoluted using the above-mentioned software. Scale bar, 5 μm (q).

Article Snippet: Cells were simultaneously stained with 1 μ m calcein AM and 2 μ m ethidium homodimer (EthD-1) in serum-free medium using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes).

Techniques: Over Expression, Staining, Immunofluorescence, Software