Computational protocol: Allosteric Modulation of Beta1 Integrin Function Induces Lung Tissue Repair

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Protocol publication

[…] Adult human lung fibroblasts (CCD-8Lu) were seeded onto collagen-I-coated culture dishes. The following day, NCI-H441 cells were seeded on top of the fibroblasts at the same density. Cells were starved with media containing 0.1% FCS. The plates were subjected PPE at 0.3 U/mL alone or in combination with JB1a (1 μg/mL) or K20 (1 μg/mL) for 1 and 3 hours. At the end of the experiment, media was aspirated and cell layer extracted using MEM-PER protein extraction kit (Pierce), protein assayed in the membrane fraction using the BCA methods, and 50 μg separated onto 10% SDS-PAGE, transferred onto Hybond-ECL (GE Healthcare). All membranes were stained with Ponceau S (Sigma) to assess the quality of the transfer and loading and probed for β1 integrin (JB1a) followed by HRP-labelled secondary antibody and developed using ECL-Plus (GE Healthcare) and exposed to Hyperfilm ECL (GE Healthcare). Densitometric analyses was carried out using ImageJ (NIH). [...] Cells were cultured as described in methods at 50,000 cell/membrane onto collagen I Bioflex membranes using silicone gaskets of 10 mm diameter. Cells were starved with media containing 0.1% FCS and Syto 16 (Molecular Probes). The media was removed, and Alexa-Fluor 647 labelled G-actin (100 μg/membrane) from rabbit was loaded using Influx (Molecular Probes). The cells were loaded with PhiPhiLux-G2D2 for visualisation of caspase activation (OncoImmune). The membrane was then mounted onto the StageFlexer (FlexCell), placed on the stage of an upright Leica-TCS-NT confocal microscope system (Leica Microsystems GmbH, Heidelberg, Germany), and subjected to 2–10% cyclic stretch at 1 Hz for up to 6 hours. Images were collected simultaneously from 3 channels at 1-minute intervals, using the ×10 lens. The resulting time-lapse movies were collated and analysed with Imaris software (Bitplane AG, Switzerland). At various time points during the study, the membrane was held static while serial optical sections were acquired the three fluorescent channels supplemented by the collection of the brightfield channel image. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Neurodegenerative Diseases, Lung Injury