/image%2F4304447%2F20200218%2Fob_ff2aa2_a27178d5-edit-img-facebook-post-image.jpg "colocalizer pro")
The absence of PI staining along with Annexin V staining of the plasma membrane at 2 hr is consistent with apoptotic, rather than necrotic, cell death. Two hours after irradiation (left), cells were treated with Cy5-Annexin V and PI and imaged. (C) Analysis of mechanism of cell death using confocal microscopy. To assess the effect of an apoptosis inhibitor, cells were incubated with 50 μM zVAD-FMK during treatment and recovery.
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Values shown in each quadrant correspond to percentages of cells observed in one experimental trial results consistent with these were obtained in three independent trials. Treated cells were incubated with Cy5-Annexin V and propidium iodide (PI) and analyzed by flow cytometry. (B) Measurement of apoptotic cell death caused by ROS generated by TO-RrRK and TO-FrFK by flow cytometry. Samples were irradiated for 15 min and then incubated and analyzed as above. Inset: comparison of the phototoxicity of TO-RrRK (gray) and TO-FrFK (black) in HeLa and MRC-5 cells. Cells were not affected by the presence of TO-FrFK or TO-RrRK alone, or light exposure alone. The amount of NADPH reduction of CCK-8 was measured at 450 nm. Cells were allowed to recover for 13 hr after irradiation, and then incubated with CCK-8 dye for 2 hr. Chemistry & Biology, DOI: ( /j.chembiol ) Copyright © 2007 Elsevier Ltd Terms and Conditionsģ Figure 2 Toxicity and Mechanism of Cell Death Characterizing Nuclear and Mitochondrial Oxidative Stress (A) Measurement of cytotoxicity using the CCK-8 assay for HeLa cells subjected to incubation with either 5 μM TO-FrFK or TO-RrRK followed by irradiation for 0–30 min. See the Supplemental Data for additional experimental data concerning localization of the conjugates. (F) Colocalization of TO-FrFK (red) with mitotracker (green) in MRC-5 cells. (E) Colocalization of TO-RrRK (red) with mitotracker (green) in MRC-5 cells.
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Quantitation of overlap using the CoLocalizer Pro software package produced a Pearson's coefficient of ± The scale bar represents 5 μm. (D) Colocalization of TO-FrFK (red) with deep red mitotracker 633 (green). Quantitation of nuclear overlap using the CoLocalizer Pro software package produced a Pearson's coefficient of ± The scale bar represents 5 μm. (C) Colocalization of TO-RrRK (red) with Syto 60 (green). The localization patterns were highly reproducible and were observed in >20 separate trials (see the Supplemental Data for additional images). Illumination conditions were optimized such that cell viability and integrity were maintained, with laser power confined to levels where no toxicity would be observed. The conditions listed above also apply to this experiment. Fluorescence image (left) is shown alongside corresponding DIC image (right). (B) Structure and fluorescence confocal microscopy images for TO-FrFK demonstrating localization of this compound within the mitochondria of living, unfixed HeLa cells. Images were acquired after incubating cells with 5 μM conjugate for 1.5 hr. Kelley Chemistry & Biology Volume 14, Issue 8, Pages (August 2007) DOI: /j.chembiol Copyright © 2007 Elsevier Ltd Terms and ConditionsĢ Figure 1 Structures and Localization Profiles for Peptidoconjugate Oxidants (A) Structure and fluorescence confocal microscopy images for TO-RrRK demonstrating localization of this compound within the nuclei of living, unfixed HeLa cells. 1 Volume 14, Issue 8, Pages 923-930 (August 2007)ĭeconvolution of the Cellular Oxidative Stress Response with Organelle-Specific Peptide Conjugates Kerry P.
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