Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Image cytometry analysis strategy of irradiated A375:NHEM co-cultures. Upon labelling with antibodies against p-γ-H2AX (red) and with Hoechst for nuclear DNA (blue), samples were automatically scanned using the TissueFAXSiPlus imaging system. After regions of interest (ROIs) reconstitution out of stitched individual fields of view (FOV), the obtained images were imported into the TissueQuest image quantification software. Each cell was identified based on nuclear DAPI signal by the segmentation algorithm. Next, scattergrams were generated based on DAPI-Eccentricity versus DAPI-Mean Intensity profiles and cell lines differentiated based on nuclear elongation (eccentricity): elongated nuclei of NHEM were gated (red gate) based on higher eccentricity values, while non-elongated A375 nuclei were gated (blue gate) based on low eccentricity values. Using forward gating, one can identify the scattergram displayed signals for each cell in a population. Here, we exemplify a NHEM nucleus encircled in red and an A375 nucleus encircled in green and their corresponding signals in the scattergrams (high vs. low elongation). In order to quantify p-γ-H2AX mean fluorescence intensity (MFI) and number of foci per nucleus, TxRed signals from the nuclear mask were quantified using 2 parameters: Texa-Mean Intensity and Texa_DOTS_ON_DAPI-Dots Count, respectively, visible on the axes. Histograms displaying frequency distribution of number of foci per nucleus are displayed in blue for each cell population. Also, scattergrams of number of foci versus p-γ-H2AX expression level can be generated. Here, red bars and dots identify the selected nuclei in the picture. Foci are indicated by white arrows (in NHEM) or arrowheads (in A375).

Article Snippet: The signals recorded through the DAPI (λ ex 360 nm/λ em 462 nm) and TxRed (λ ex 568 nm/ λ em 603 nm) filters were further analysed upon nuclear segmentation using TissueQuest software version n. 4.0.1.0140 (TissueGnostics, Vienna, Austria, URL: https://tissuegnostics.com/products/single-cell-analysis/tissuequest ,).

Techniques: Cytometry, Irradiation, Imaging, Software, Generated, Fluorescence, Expressing

Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Analysis of the A375 melanoma and NHEM co-cultures upon laser-plasma accelerated electron exposure during 2 sequentially performed irradiation experiments (PW1, PW2) versus pulsed X-ray irradiation. ( a , c ) Correspondence between the irradiated GF fingerprint and selected areas of the SlideFlasks for image cytometry analysis (5 × magnification preview mode). ( b , d , e , f ) DNA damage response revealed as p-γ-H2AX foci at 30 min post-exposure to PW or X-ray-generated radiation versus non-exposed (untreated) or mock-exposed (PW env) negative controls. Immunofluorescence microscopy images of overlapping DAPI and TxRed signals (top), as well as grayscale images of the TxRed signal (bottom) of a representative field of view (FOV) under each irradiation condition, are shown. The p-γ-H2AX foci are visible in the detailed insets ( b , d , f —bottom right). Scale bar = 20 μm ( b , d , f ) or 100 μm ( e ).

Article Snippet: The signals recorded through the DAPI (λ ex 360 nm/λ em 462 nm) and TxRed (λ ex 568 nm/ λ em 603 nm) filters were further analysed upon nuclear segmentation using TissueQuest software version n. 4.0.1.0140 (TissueGnostics, Vienna, Austria, URL: https://tissuegnostics.com/products/single-cell-analysis/tissuequest ,).

Techniques: Clinical Proteomics, Irradiation, Cytometry, Generated, Immunofluorescence, Microscopy