Journal: Radiation Oncology (London, England)

Article Title: The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage

doi: 10.1186/s13014-015-0453-1

Figure Lengend Snippet: The ImageJ-based interface of the Focinator offers options to adapt the evaluation parameters to distinct image characteristics. Figure 1 shows ImageJ with the Focinator macro installed as start-up macro after opening a multi-channel image. This microscope image with the file format ZVI 16-bit includes three fluorescence channels. The main window of the Focinator is implemented into the ImageJ window. It consists of a menu ( 2 ), buttons ( 1 ) and Focinator Options ( 3 and 4 ). The Focinator Options windows offer several preferences for the user to adapt the macro’s behavior to individual requirements. Picture Settings: First step is to tell the macro, the input folder and if there is a multi-channel image or more single pictures will be opened. In the second step you choose in which channel the foci have to be counted and where the ROIs should be selected. In our example, the γ-H2.AX foci are in channel number 2 ( on top after opening the image ). The macro will use the setting “1st foci channel = front channel” for all pictures automatically. If no second foci channel is used the setting should be changed to “inactive”. ROI Settings ( 3 ): Depending on image quality, size and magnification, it is recommended to set the threshold and the size filters for ROIs. Alternatively, the choice of automated thresholding is possible. It is possible to exclude objects that are partially outside of the image. If there are objects to exclude because they are not circular enough or too small, it is possible to exclude them via circularity filters or size filters. “Use fill holes” should be activated, if the ROI selection left holes in the cells. Overlapping ROIs (cells, nuclei) might be separated by choosing “watershed”. Regarding the batch mode “check selection” offers the possibility of stopping during the selection process. “Invert images” should be checked when working with images with light background. For the automated batch ( 4 ) mode, output directories need to be chosen to save the results. An important step of evaluation is to choose the right noise level. Noise level values can be set independently in multi-channel analysis to exclude background artifacts. By defining the cut off, foci with intensities below a certain value are deleted, which excludes background noise. The value for area correction is dependent on the mean size of the analyzed nuclei. The factor corrects the foci number divided by the individual area of each nucleus. The usage of the percentile option enables the user to delete the outliers, such as cells with false γ-H2.AX foci induced by replication. Colocalization analyses are also possible. This option compares the localization of two foci in two different channels with a selectable tolerance

Article Snippet: Antibodies linked with Alexa Fluor 647 against γ-H2.AX protein were obtained from Becton Dickinson (Heidelberg, Germany).

Techniques: Microscopy, Fluorescence, Selection

Journal: Radiation Oncology (London, England)

Article Title: The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage

doi: 10.1186/s13014-015-0453-1

Figure Lengend Snippet: Use of the Focinator macro reduces counting times compared to ImageJ-based counting and manual evaluation. TRAMP-C1 cells were irradiated with 3 Gy. The cells were fixed and permeabilized for 15 min with 3 % PFA and 0.2 % Triton X-100 at different time points after irradiation. The nuclei were stained with Hoechst 33342. DSB foci were labeled with Alexa Fluor 647-linked anti- γ-H2.AX antibodies. The evaluation time for the same 35 multi-channel images containing 439 nuclei was compared between the analysis with the Focinator, ImageJ-based counting via manual ROI marking and “ Find Maxima… ” function or manual counting. a Evaluation times using the different counting methods. b Comparison of detected nuclei numbers by ImageJ-based analysis, Focinator batch mode and manual counting shown as overall ROI count

Article Snippet: Antibodies linked with Alexa Fluor 647 against γ-H2.AX protein were obtained from Becton Dickinson (Heidelberg, Germany).

Techniques: Irradiation, Staining, Labeling

Journal: Radiation Oncology (London, England)

Article Title: The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage

doi: 10.1186/s13014-015-0453-1

Figure Lengend Snippet: The Focinator’s accuracy is comparable to manual counting and evaluation only with ImageJ. ImageJ-based, manual counting and the usage of the Focinator macro were compared. To evaluate the repair time-dependent decrease of γ-H2.AX foci after irradiation TRAMP-C1 cells were irradiated with 3 Gy, incubated at 37 °C and fixed 0.5, 1, 2, 4, 6 and 24 h after irradiation. The cells were permeabilized and stained with an Alexa 647-linked anti- γ-H2.AX antibody. A total number of approximately 40 nuclei per time point was evaluated. a Development of the mean foci count per nucleus form three independent experiments at stated time points after irradiation. b A dose response curve depicts foci count after different doses (0.5, 1.5 and 3 Gy) 30 min after irradiation. A direct correlation between the different scoring methods with respective correlations value (R 2 ) at the time points 0.5, 1, 2, 4, 6 and 24 h after irradiation is shown for Focinator-based evaluation in comparison to using ImageJ alone in ( c ) and compared to manual counting in ( d )

Article Snippet: Antibodies linked with Alexa Fluor 647 against γ-H2.AX protein were obtained from Becton Dickinson (Heidelberg, Germany).

Techniques: Irradiation, Incubation, Staining

Journal: bioRxiv

Article Title: The interaction of aging and oxidative stress contributes to pathogenesis in mouse and human Huntington disease neurons

doi: 10.1101/800268

Figure Lengend Snippet: Primary Hu18/18 and Hu97/18 neurons were treated with NBC vehicle or 100µM H 2 O 2 for 24h. ( A-D ) DNA damage was assessed by 8-Oxo-dG immunocytochemistry. ( A,B ) Representative images ( A ) and quantification ( B ) of % 8-Oxo-dG positive immature neurons. ( C,D ) Representative images ( C ) and quantification ( D ) of % 8-Oxo-dG positive mature neurons. ( E,F ) Cell death was assessed by quantifying LDH released/total in ( E ) immature and ( F ) mature neurons. *=difference between indicated bars, *p<0.05, **p<0.01, ****=p<0.0001. Error bars ± SEM.

Article Snippet: The second set was stained for a marker of apoptosis (cleaved caspase-3), DNA damage (γ-H 2 AX 1:1000, Upstate Biotechnology), and viral transduction (GFP 1:2000, Abcam ab13970).

Techniques: Immunocytochemistry

Journal: bioRxiv

Article Title: The interaction of aging and oxidative stress contributes to pathogenesis in mouse and human Huntington disease neurons

doi: 10.1101/800268

Figure Lengend Snippet: ( A ) Mature Hu18/18 and Hu97/18 neurons were treated with nGFP or progerin to induce aging, and mtHTT was assessed by EM48 immunocytochemistry. ( B,C ) Mature Hu18/18 and Hu97/18 neurons were aged with progerin or given nGFP control followed by H 2 O 2 or vehicle control treatment. Oxidative DNA damage was assessed by oxo-8-dG immunocytochemistry. Representative images ( B ) and quantitation of % of neurons exhibiting DNA damage ( C ).( D ) Mature primary Hu18/18 and Hu97/18 neurons were treated with nGFP or progerin followed by H 2 O 2 or vehicle control. Cell death was assessed by quantifying released/total LDH. *=difference between indicated bars, *=p<0.05, **p<0.01, ****=p<0.0001. Error bars ± SEM.

Article Snippet: The second set was stained for a marker of apoptosis (cleaved caspase-3), DNA damage (γ-H 2 AX 1:1000, Upstate Biotechnology), and viral transduction (GFP 1:2000, Abcam ab13970).

Techniques: Immunocytochemistry, Control, Quantitation Assay

Journal: bioRxiv

Article Title: The interaction of aging and oxidative stress contributes to pathogenesis in mouse and human Huntington disease neurons

doi: 10.1101/800268

Figure Lengend Snippet: HD patient and healthy control iPSCs were differentiated toward striatal-like neurons and treated with nGFP or progerin and evaluated by immunocytochemistry. ( A ) GFP staining demonstrates nuclear blebbing in progerin, but not nGFP treated neurons. ( B-D ) Dendritic morphology was assessed in transduced cells by ( C ) total dendrite length and ( D ) radial dendritic complexity. ( E ) Apoptosis was assessed by % of transduced neurons that are caspase-3 positive. ( F ) DNA damage was assessed by % of transduced neurons with >3 γH 2 AX foci, revealing a selective increase in HD neurons. *=difference between indicated bars. *p<0.05. Error bars ± SEM.

Article Snippet: The second set was stained for a marker of apoptosis (cleaved caspase-3), DNA damage (γ-H 2 AX 1:1000, Upstate Biotechnology), and viral transduction (GFP 1:2000, Abcam ab13970).

Techniques: Control, Immunocytochemistry, Staining

Journal: bioRxiv

Article Title: The interaction of aging and oxidative stress contributes to pathogenesis in mouse and human Huntington disease neurons

doi: 10.1101/800268

Figure Lengend Snippet: ( 1 ) wtHTT is a stress response protein that can form Huntingtin stress bodies in the cytoplasm , can act as a ROS sensor , and aid in the base excision repair pathway in the nucleus in response to DNA damage . ( 2 ) Aging increases oxidative stress in cells ( ; ). ( 3 ) Aging increases DNA damage . ( 4 ) Oxidative stress increases oxidative damage, . ( 5 ) DNA damage increases DNA repair particularly the base-excision repair pathway ( ; ; ). ( 6 ) mtHTT does not perform its stress response functions in the cell as well ( ; ). Additionally, ( 7 ) accelerated epigenetic aging has been found in age-matched HD brains and age-related nuclear pore complex deficiencies have been found in HD , which collectively drive greater age-related oxidative stress and DNA damage and greater subsequent base excision DNA repair. ( 8 ) Altering the rate of base-excision repair can increase somatic instability of the CAG tract ( ; ; ). ( 9 ) Oxidative stress can cause somatic instability of the CAG tract , possibly through the DNA damage/DNA repair pathway. ( 10 ) Somatic instability of the CAG tract is a driver of mtHTT toxicity, and may be necessary in certain tissues for onset and progression of HD ( ; ; ).

Article Snippet: The second set was stained for a marker of apoptosis (cleaved caspase-3), DNA damage (γ-H 2 AX 1:1000, Upstate Biotechnology), and viral transduction (GFP 1:2000, Abcam ab13970).

Techniques: