Computational protocol: Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drivespancreatitis

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Protocol publication

[…] Histochemical staining was performed on paraffin-embedded slides with classical haematoxylin eosin or Masson trichrome staining procedure. Immunofluorescence of cryosections or paraffin-embedded slides was performed as described below and recorded on either a confocal laser scanning microscope or a standard fluorescence microscope (Leica, Germany) using overnight hybridization with primary antibodies specific for α-smooth muscle actin (Abcam, Cambridge, UK, 1:500), cleaved Caspase 3 (Cell Signaling, NEB, 1:300), Cramp (Innovagen, Lund, Sweden, 1:200), neutrophil elastase (Abcam, 1:200), EpCAM (BioLegend, 1:100), F4/80 (eBioscience, 1:1,000), citrullinated histone H3 (Abcam, 1:200), human IL-17A (R&D Systems, Wiesbaden, Germany, 1:100), mouse IL-17A (Santa Cruz, Heidelberg, Germany, 1:500) and MPO (Abcam, 1:200). Detection was performed using either biotinylated secondary antibodies (goat anti-rabbit or anti-rat, Abcam, 1:1,000) and TSA Fluorescein/Cy3 kits (PerkinElmer, Waltham, MA, USA) or directly labelled Alexa 488 or Alexa 555-conjugated goat anti-rat antibodies (Abcam, 1:200–1:1,000). Before examination, the nuclei were counterstained with Hoechst 33342, propidium iodide or Sytox Green (Invitrogen Molecular Probes, Karlsruhe, Germany; BD, Heidelberg, Germany). TUNEL for the in situ detection of cell death was performed using the Roche in situ cell death detection kit according to the manufacturer's protocol (Roche, Mannheim, Germany). NET quantification was performed using mean fluorescence intensity and area analysis functions of Adobe Photoshop CS5. Flow cytometry was performed on a BD Fortessa instrument after surface staining of CD11b (1:500), Ly6G (1:500), CD4 (1:500), CD8 (1:500) and B220 (1:500) coupled with different standard fluorophores FITC, PE, PE-Cy7, APC and PerCP-Cy5.5 (BioLegend). Cells were gated on live cells and cell death exclusion was performed using PI (1:100) or 7AAD (1:100) staining before flow cytometry. Recording was performed using FACS DIVA software, further analysis was performed using FlowJo 7.6.5 software. [...] For the flow cytometric measurement of intracellular pH, human granulocytes were incubated with the pH indicator SNARF-1 (Life Technologies, Germany) and the fluorescent emissions at various concentrations of NaHCO3 were recorded with a Gallios Flow Cytometer (Beckman Coulter, USA). The shift from 575 to 660 nm emission was considered as an indicator of pH and was evaluated with the Beckman Coulter analysis software Kaluza 1.3. […]

Pipeline specifications

Software tools FlowJo, Kaluza
Application Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Arterial Occlusive Diseases, Pancreatic Neoplasms
Chemicals Calcium Carbonate