cometchip system  (Bio-Techne corporation)

Journal: Nature Communications

Article Title: Base excision repair and double strand break repair cooperate to modulate the formation of unrepaired double strand breaks in mouse brain

doi: 10.1038/s41467-024-51906-5

Figure Lengend Snippet: a Representative IF images of stained neurons and glia in CTX of 7 weeks C57BL/6J male mice. NeuN (purple) and γH2AX (green) prominently co-stain in neurons. Tissue staining in each cell is displayed in a series of separate channel images for DAPI (blue), NeuN (purple), or γH2AX (green), as indicated, or as an overlay of all three (D/N/H) (left). (top row) Field of stained cells for DAPI, NeuN, and γH2A-X antibodies (D/N/H) (Scale bar is 10 µm); neurons co-stained with NeuN and DAPI, while glia stained only with DAPI. Magnified images of Neuron (N) (middle row) and glia (G) (bottom row) were selected from the cell fields in the top row (Scale bar is 1 µm). b The DSB markers γH2AX (green) and 53BP1 (red) were identified as neuronal if they co-stained with NeuN (purple) in male C57BL/6J mouse tissue; γH2AX (green) and 53BP1 (red) markers in glia co-stained only with DAPI (blue). c Quantification of IF staining intensity for γH2AX from n = 50 randomly selected cells of each type in n = 3 tissue sections of 7 (left) or 75 (right) weeks animals (from a ), respectively. Data are displayed as a box and whisker plot, where the box are 25–75% of the values, the line indicates the median value, and 25% maximum values and 25% minimum values are indicated by whiskers above and below the box, respectively. The probability statistics for comparing significance among regions were determined from a one-way ANOVA are **** P < 0.0001 for all type comparisons. d Neutral CometChip assay results for dispersed cells from different brain tissues (CBL, STR, CTX, HIP) at 7 weeks and 75 weeks. e Quantification of the comet tail length (μm) in dispersed cells from ( d ) as a function of age; 7 weeks, gray; 75 weeks, black. Points shown are individual scored comets from at least n = 550–890 cells per tissue section ( n = 3 animals). The probability statistics for comparing the Neutral CometChip Tail lengths of dispersed cells at the two ages were determined from a one-way ANOVA. **** P < 0.0001 for all comparisons.

Article Snippet: All chemical reagents used for CometChip assays were obtained from Sigma or VWR unless otherwise stated.

Techniques: Staining, Whisker Assay

Journal: Nature Communications

Article Title: Base excision repair and double strand break repair cooperate to modulate the formation of unrepaired double strand breaks in mouse brain

doi: 10.1038/s41467-024-51906-5

Figure Lengend Snippet: a Representative digital images of comet tails (orange) (µm) from a neutral CometChip assay in cultured glial cells from CBL, HIP, CTX, and STR. The software delineates the features of each identified comet, as indicated by the lines at the bottom of each image. The region bounded by the white and red lines signifies the head portion of the comet, while the space between the red and the green lines illustrates the tail section. The green line extends to the fluorescence intensity distribution of the tail region, eliminating the background intensity. b Result of comet tail length (µm) for cultured glial cells from CBL (blue), HIP (orange), CTX (pink), and STR (green) of dissected tissue of n = 4 animals. Points are individual scored comets. At least n = 50 cells per regional glial line were scored in three technical replicates ( n = 3) measured on separate days. Data are displayed as a box and whisker plot, where the box are 25–75% of the values, and 25% maximum values and 25% minimum values are indicated by whiskers above and below the box, respectively. The line in the box indicates the median value. Regional comparions were determined using a one-way ANOVA. **** P < 0.00001 for all comparisons. c Parallel γH2AX staining intensity in duplicate plates of cultured glial cells from ( b ); CBL (blue), HIP (orange), CTX (pink), and STR (green) displayed as a box and whisker plot as in ( b ). Regional comparions were determined using a one-way ANOVA, * P = < 0.01, ** P < 0.001.

Article Snippet: All chemical reagents used for CometChip assays were obtained from Sigma or VWR unless otherwise stated.

Techniques: Cell Culture, Software, Fluorescence, Whisker Assay, Staining

Journal: Nature Communications

Article Title: Base excision repair and double strand break repair cooperate to modulate the formation of unrepaired double strand breaks in mouse brain

doi: 10.1038/s41467-024-51906-5

Figure Lengend Snippet: a Schematic illustration of a transient SSB intermediate generated by excision of oxidized base damage during BER. b Schematic diagram of the 8-oxo-G ELISA assay steps. c , d Quantification of 8-oxo-G in cells from mouse brain ( n = 3) in four regions, CBL (blue), STR (green), CTX (pink), and HIP (orange) at 7 ( c ) and 75 ( d ) weeks using the competitive ELISA assay (Cayman Chemical). Data are displayed as a box and whisker plot, where the line indicates the median value, the box are 25–75% of the values, and 25% maximum values and 25% minimum values are indicated by whiskers above and below the box, respectively. The probability statistics for regional comparisons was determined from a one-way ANOVA, * P < 0.01, ** P < 0.001, *** P < 0.0001. e n = 4 technical replicates of Alkaline CometChip images illustrate the presence of SSBs in dispersed cells from dissected brain regions (CBL, STR, CTX, HIP) of 7 or 75 weeks animals. f Analysis of the comet tail length for ( e ) at 7 weeks (gray) and 75 weeks (black). Each symbol represents an individual scored comet ( n > 700). SSBs increase with age in all brain regions. Data are displayed as a box and whisker plot as defined in ( c , d ). Source data are provided in Source data file. The probability statistics for age comparisons in each region was determined from a one-way ANOVA. **** P < 0.00001.

Article Snippet: All chemical reagents used for CometChip assays were obtained from Sigma or VWR unless otherwise stated.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Whisker Assay

Journal: NAR Cancer

Article Title: Alkylation of nucleobases by 2-chloro- N,N -diethylethanamine hydrochloride (CDEAH) sensitizes PARP1 -deficient tumors

doi: 10.1093/narcan/zcad042

Figure Lengend Snippet: CDEAH induces more DNA double-strand breaks (DSBs) in PARP1-deficient cells. ( A ) Cell cycle of HCT116 after CDEAH treatment. HCT116 WT and PARP1-deficient cells were incubated with different doses of CDEAH for 24 h and the relative percentage of cell cycle stages was calculated by FlowJo software. ( B ) DSB occurrence caused by CDEAH treatment was confirmed by γ-H2AX. HCT116 WT or PARP1-deficient cells were incubated with 80 μM CDEAH for 24 h and indicated protein level was determined in whole-cell extracts. ( C ) CDEAH treatment enhances DNA damage in HCT116 PARP1-deficient cells. The tail moment in the CometChip ® assay was calculated using the Comet analysis software (Trevigen). ( D ) SCE analysis in HCT116 WT and PARP1-deficient cells. SCEs were imaged by a BX53 microscope. At least 20 metaphases per each condition were analyzed. ( E ) Abnormal chromosomes were analyzed in HCT116 WT and PARP1-deficient cells. Abnormal chromosomes were imaged by a BX53 microscope. At least 20 metaphases per each condition were analyzed. ( F ) Graph displaying the number of SCEs in one metaphase in different conditions. ( G ) Cells that have >25 breaks in one metaphase were analyzed from panel (E). ( H ) CDEAH treatment causes more apoptotic cell death in PARP1-deficient cells. Apoptotic cell death was quantified using an Annexin V Alexa Fluor™ 488 conjugate and analyzed by flow cytometry. Data are presented as mean ± SEM.

Article Snippet: The comet assay was performed using a CometChip ® (Trevigen) according to the manufacturer’s instructions.

Techniques: Incubation, Software, Microscopy, Flow Cytometry