Computational protocol: TRPV4 inhibition attenuates stretch-induced inflammatory cellular responses and lung barrier dysfunction during mechanical ventilation

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Protocol publication

[…] Uniaxial cell strain was performed on the Stretch/compression device (University Ulm, Ulm, Germany), a device for simultaneous live cell imaging during uni-axial mechanical strain or compression []. An elastic silicon membrane (Specialty Manufacturing, Saginaw, MI 48603–3440 USA) was cut into a rectangular piece (9x2cm) and clamped into the membrane holders that shape the membrane into a chamber (see Stretch/compression device) [], autoclaved and coated overnight at 4°C with fibronectin (5μg/ml in PBS, both from Sigma-Aldrich, Steinheim, Germany). Human lung epithelial cells (NCI-H292) were seeded in the elastic silicon chamber (4x105/membrane) and cultivated in medium at 37° C in 5% CO2, humidified air for 24h. Prior to imaging, the cells were pre-incubated in medium at 37°C/5% CO2 with 2μM of the fluorescent Ca2+ dye fluo-4 and 0.2% Pluronic F127 (Molecular Probes; Karlsruhe, Germany), protected from light with or without the TRPV4 antagonist GSK2193874 (1μM) for 30 min and another 30 min at RT. For cell stretch, the medium was replaced with bath solution (pH 7.4; 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 10 mM HEPES; all from Sigma-Aldrich). Then the membranes were fastened onto the stretch apparatus [], mounted on a Zeiss Axiovert 200 (Carl Zeiss, Oberkochen, Germany) with a 20X plan Neofluar Zeiss objective. Images were acquired with a CoolSnap EZ CCD camera and Metamorph software (exposure time of 30ms and an acquisition rate of 0.5 frames per second) and an EGFPfilter cube (excitation 470/20 nm, emission 525/25 nm, dichroic 490 nm). The membranes were stretched at RT with a triangular waveform one single time from 0% to 80% length increase and back to 0% within 800 ms.The average grey values in the image sequence were determined with ImageJ [] by drawing a region of interest that comprised the adherence area of a single cell. To compensate for the slight sideward shift of the cell after the strain, the region of interest was manually repositioned. Data were transferred to MS-excel and after background subtraction, the average fluorescence values of each cell before and 10 s after the strain were determined. The strain-induced change after stimulation was expressed as the % change intensity compared to baseline signal before stretch. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications SPIM, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Ventilator-Induced Lung Injury
Chemicals Calcium, Ruthenium