Journal: BioMed Research International

Article Title: Excess Hydrocortisone Hampers Placental Nutrient Uptake Disrupting Cellular Metabolism

doi: 10.1155/2018/5106174

Figure Lengend Snippet: Assessment of hydrocortisone-induced cellular death by apoptosis in healthy-term placental explants. (a) Representative microphotographs of TUNEL assay to determine apoptosis in each experimental condition; cells undergoing apoptosis present fluorescent green nuclei. (b) Quantification of the % of TUNEL positive cells per condition. (c) Active caspases 3 and 7 as measured by luminometry, using the Caspase Glo Assay. (d) Cleaved caspase 3 as determined by western blot and normalized by actin levels. Values are presented as means ± SEM for 5 independent experiments. ∗ p≤0.05; ∗∗ p≤0.01; ∗∗∗ p≤0.001 relative to control.

Article Snippet: Apoptosis was assessed with a commercially available fluorescent terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) kit (Dead-End TM Fluorometric TUNEL System Kit; Promega, Madison, WI, USA).

Techniques: TUNEL Assay, Caspase-Glo Assay, Western Blot, Control

Journal:

Article Title: Oxidative Damage and Defective DNA Repair is Linked to Apoptosis of Migrating Neurons and Progenitors During Cerebral Cortex Development in Ku70-Deficient Mice

doi: 10.1093/cercor/bhh171

Figure Lengend Snippet: Ku70−/− cerebral cortex exhibits clustered cell death. (A, B) E14.5 Ku70−/− cortex stained with SYTOX Green. Prominent clusters of pyknotic cells were observed in Ku70 −/− mice, but not heterozygous littermates; the inset in (A) is a magnified view of a cluster of pyknotic nuclei in the Ku70−/− cortex; the inset in (B) shows a magnified view of a pyknotic nucleus in the Ku70+/− cortex; (C, D) a striking increase in TUNEL staining was observed in the Ku70−/− cortex compared with Ku70+/− cortex; the inset in (C) shows a magnified view of a cluster of TUNEL-positive cells; (E, F) co-localization of TUNEL (green) and active caspase-3 (red) indicated caspase-3 activation, suggesting an apoptotic form of cell death; the inset in (E) shows a magnified view of dual-labeled clusters of dying cells in a Ku70−/− cortex showing TUNEL and active caspase-3 staining. Arrows point to apoptotic cells that are distributed throughout the ventricular zone (vz), intermediate zone (iz), and developing cortical plate (cp). Arrowheads point to clusters of apoptotic cells. Scale bar in large panels = 100 μm; in insets = 10 μm.

Article Snippet: TUNEL Staining Fluorescent terminal dUTP nick end labeling (TUNEL; Serologicals Corporation, Norcross, GA) was used to detect the presence of DNA strand breaks in the tissue sections, following manufacturer's instructions for frozen sections.

Techniques: Staining, TUNEL Assay, Activation Assay, Labeling

Journal:

Article Title: Oxidative Damage and Defective DNA Repair is Linked to Apoptosis of Migrating Neurons and Progenitors During Cerebral Cortex Development in Ku70-Deficient Mice

doi: 10.1093/cercor/bhh171

Figure Lengend Snippet: Ku70−/− mice exhibit elevated cell death from E14.5 through P0, but comparable levels of death at P4 and P8. (A) Scatter plot representing quantification of cell death by TUNEL at different ages starting at E14.5 through P8; (B) quantification of cell death by pyknosis from E14.5 to P8; C, quantification of cell death by immunostaining for activated caspase-3 from E14.5 to P8. Solid squares indicate Ku70−/− mice, gray triangles represent Ku70+/− mice, and hollow circles are Ku70+/+ mice. Asterisks indicate statistical significance by two-tailed t-test, P < 0.0001. Error bars indicate standard error of the mean. Each data point represents counts for one mouse.

Article Snippet: TUNEL Staining Fluorescent terminal dUTP nick end labeling (TUNEL; Serologicals Corporation, Norcross, GA) was used to detect the presence of DNA strand breaks in the tissue sections, following manufacturer's instructions for frozen sections.

Techniques: TUNEL Assay, Immunostaining, Two Tailed Test

Journal:

Article Title: Oxidative Damage and Defective DNA Repair is Linked to Apoptosis of Migrating Neurons and Progenitors During Cerebral Cortex Development in Ku70-Deficient Mice

doi: 10.1093/cercor/bhh171

Figure Lengend Snippet: Apoptotic cells in Ku70−/− embryonic cortex co-label with markers for radial glia and migrating, immature neurons. (A–C) cells dying in G1–S phase co-labeled with TUNEL (green) and Ki67 (red); (D–F) a cluster of dying cells in the S phase, located in the cortical intermediate zone, demonstrated by co-labeling with TUNEL (green) and PCNA (red); (G–I) dying radial glia showed co-labeling for TUNEL (green) and GLAST (red); (J–L) neurons dying during migration were co-labeled by TUNEL (green) and doublecortin (red); (M–O) dying post-mitotic neurons showed co-labeling for TUNEL (green) and β3-tubulin (red). Dual labeled cells for Ki67, PCNA and GLAST appear yellow in merged images (arrows); DCX and β3-tubulin are non-nuclear. Therefore, co-labeling is inferred from co-expression in the same focal plane. Scale bar = 10 μM.

Article Snippet: TUNEL Staining Fluorescent terminal dUTP nick end labeling (TUNEL; Serologicals Corporation, Norcross, GA) was used to detect the presence of DNA strand breaks in the tissue sections, following manufacturer's instructions for frozen sections.

Techniques: Labeling, TUNEL Assay, Migration, Expressing

Journal:

Article Title: Oxidative Damage and Defective DNA Repair is Linked to Apoptosis of Migrating Neurons and Progenitors During Cerebral Cortex Development in Ku70-Deficient Mice

doi: 10.1093/cercor/bhh171

Figure Lengend Snippet: Quantification of apoptotic cells in Ku70−/− mice. (A) Co-labeling of TUNEL with various cell cycle markers at E14.5 showed a significant increase in the number of dying cells in the G1 (Ki67) and S (Ki67 and PCNA) phases of the cell cycle; (B) co-labeling of TUNEL and different cell type-specific markers showed a significant increase in radial glia (GLAST) and immature neurons (β3-tubulin and DCX); (C) co-labeling of TUNEL with cell cycle markers at P0 indicated no differences in the G2–M phase (pH3), but increased cell death in G1 and S phases of the cell cycle (Ki67 ); (D) co-labeling with cell type-specific markers showed increased radial glial and immature neuron death. Most of the TUNEL-positive cells expressed one or more of the markers examined, as shown by the TUNEL-cocktail co-labeling. Solid black bars represent Ku70−/− mice and hatched bars represent Ku70+/− mice in the graphs. Asterisks indicate statistical significance by two-tailed t-test, P < 0.0001. Error bars indicate standard error of the mean.

Article Snippet: TUNEL Staining Fluorescent terminal dUTP nick end labeling (TUNEL; Serologicals Corporation, Norcross, GA) was used to detect the presence of DNA strand breaks in the tissue sections, following manufacturer's instructions for frozen sections.

Techniques: Labeling, TUNEL Assay, Two Tailed Test

Journal:

Article Title: Oxidative Damage and Defective DNA Repair is Linked to Apoptosis of Migrating Neurons and Progenitors During Cerebral Cortex Development in Ku70-Deficient Mice

doi: 10.1093/cercor/bhh171

Figure Lengend Snippet: Ku70−/− brains exhibit increased oxidative damage; lowering oxygen tension rescues Ku70−/− and Ku70+/− neurons from apoptosis. (A) perinuclear 8-OHdG immunostaining was observed in the embryonic cerebral cortex of Ku70−/− mice; (B) 8-OHdG immunostaining was rare in the cortices from heterozygous mice at this age; (C) high oxygen (20%) resulted in increased pyknosis in the neuronal cultures; (D) higher oxygen levels were associated with significant increases in TUNEL-positive nuclei; (E) Tempol treatment of Ku70+/− and Ku70−/− cultures reduced cell death to wildtype levels at both 20 and 3% oxygen as assessed by pyknosis; (F) Tempol treatment reduced numbers of TUNEL+ cells in Ku70+/− and Ku70−/− cultures to wildtype levels at both 20 and 3% oxygen. Solid black bars represent Ku70−/− mice, grey bars Ku70+/−, and empty bars Ku70+/+ mice. Asterisks indicate statistical significance by two-tailed t-test, P < 0.0001. Error bars indicate standard error of the mean. Scale bar in large panels = 100 μm; in inset = 15 μm.

Article Snippet: TUNEL Staining Fluorescent terminal dUTP nick end labeling (TUNEL; Serologicals Corporation, Norcross, GA) was used to detect the presence of DNA strand breaks in the tissue sections, following manufacturer's instructions for frozen sections.

Techniques: Immunostaining, TUNEL Assay, Two Tailed Test