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

Journal: Scientific Reports

Article Title: A minimalist model to measure interactions between proteins and synaptic vesicles

doi: 10.1038/s41598-020-77887-1

Figure Lengend Snippet: Patterning strategy and SV immobilization. ( a ) Schematic representation of photopatterning of neutravidin on a glass coverslip. The glass coverslip was uniformly coated with an anti-fouling layer of PLL-g-PEG. The photoinitiator (PLPP) was added on top of the PLL-g-PEG layer and the substrate was exposed to UV light through a virtual photomask to activate the PLPP. Under UV light, the PLPP degraded the PLL-PEG layer, leaving accessible regions for neutravidin to attach. ( b ) Schematic of our strategy to attach the SVs to the substrate. The purified SVs (gray) were attached to the neutravidin (pink) functionalized glass coverslips via a biotinylated mouse anti-synaptotagmin antibody (orange). The attachment of antibodies was controlled by a secondary anti-mouse antibody labeled with Alexa Fluor 488 (green). To image the SVs, a single-domain antibody against vGLUT1, labeled with STAR635P (red) was employed. ( c ) Fluorescence micrographs of the individual steps of the vesicle immobilization strategy. The neutravidin functionalization step was assessed by fluorescently labeled neutravidin (neutravidin-FITC, left), the attachment of the biotinylated mouse anti-synaptotagmin antibody was tested with a secondary anti-mouse antibody labeled with Alexa Fluor 488 (center), and the SV attachment was tested with a single-domain antibody against vGLUT1 labeled with STAR635P (right). Note that the uniform fluorescence in the right image shows the bound SVs, whereas the small bright spots are aggregates. The images of neutravidin-FITC and anti-ms-Alexa488 were taken on two different glass coverslips. In the actual experiments, we used unlabeled neutravidin, so as to not interfere with the fluorescence of the mEGFP-tagged proteins. The scale bars are 25 μm.

Article Snippet: The excitation light came from a mercury arc lamp (X-Cite 120 PC Q, Excelitas Technologies, Waltham, USA) and was guided onto a fluorescence filter cube (filter sets available: DAPI, GFP, Cy3, TxRed and Cy5, all from AHF Analysentechnik AG).

Techniques: Purification, Labeling, Fluorescence

Journal: Scientific Reports

Article Title: A minimalist model to measure interactions between proteins and synaptic vesicles

doi: 10.1038/s41598-020-77887-1

Figure Lengend Snippet: Comparison of cell lysate and purified α-synuclein-mEGFP. ( a ) Average normalized ACF of purified α-synuclein-mEGFP (closed squares, N = 30) and α-synuclein-mEGFP from HEK cell lysate (open squares, N = 30); there was no SV pattern present in these experiments; both protein preparations show the same diffusion behavior. ( b ) The purified α-synuclein-mEGFP interacted with the SV pattern (left hand side), as shown by the fluorescence signal (right; SV pattern visualized by anti-vGlut-STAR635P). ( c ) By contrast, α-synuclein-mEGFP from the cell lysate (left) did not bind to the SV pattern (right). The images in ( b ) and ( c ) were recorded after 2 hours of incubation of the SV layer with α-synuclein-mEGFP. The scale bars are 25 μm. Note that the uniform fluorescence in the right images in ( b ) and ( c ) shows the bound SVs, whereas the small bright spots are aggregates.

Article Snippet: The excitation light came from a mercury arc lamp (X-Cite 120 PC Q, Excelitas Technologies, Waltham, USA) and was guided onto a fluorescence filter cube (filter sets available: DAPI, GFP, Cy3, TxRed and Cy5, all from AHF Analysentechnik AG).

Techniques: Comparison, Purification, Diffusion-based Assay, Fluorescence, Incubation