Computational protocol: Inflammation Induces Irreversible Biophysical Changes in Isolated Nucleus Pulposus Cells

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Protocol publication

[…] Expression of cell cytoskeletal elements (F-actin and β-tubulin) and of the water channel aquaporin-1 was analyzed. Cells from each treatment and recovery group were plated on poly-lysine coated coverslips and allowed to adhere for 30 minutes (to maintain round morphology as in osmotic loading experiments). For F-actin staining, cells were fixed in 4% paraformaldehyde, permeabilized for 5 minutes with 1% Triton-X 100, and incubated for 30 minutes at room temperature with rhodamine phalloidin (1∶100 dilution, Molecular Probes, Eugene, OR, USA). For β-tubulin staining, cells were fixed in 0.3% glutaraldehyde, permeabilized for 15 minutes with 1% Trition-X 100, and incubated for 90 minutes at 37°C with a mouse anti-β-tubulin primary antibody (1∶200 dilution, Sigma-Aldrich, St. Louis, MO, USA) followed by incubation with Alexa Fluor 488 goat anti-mouse secondary antibody for 60 minutes at 37°C (1∶200 dilution, Molecular Probes, Eugene, OR, USA). Cells were imaged with confocal microscopy (Fluoview 300, Olympus, Center Valley, PA, USA) at 60x magnification and optical slices were taken at 2 µm intervals throughout the cross section. Images of cytoskeletal structure captured at the cell’s midplane are presented.For aquaporin-1 expression, cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100, and incubated overnight at 4°C with a rabbit anti-aquaporin-1 primary antibody (1∶200 dilution, Abcam, Cambridge, MA, USA) followed by incubation with an Alexa Fluor 488 goat anti-rabbit secondary antibody for 60 minutes at room temperature (1∶200 dilution, Molecular Probes, Eugene, OR, USA). Cells were imaged via fluorescence microscope (Axiovert 200 M, Zeiss, Thornwood, NY, USA) at 60x magnification using constant exposure time for each image. Quantification of Aqp-1 expression was performed by analyzing cells in ImageJ software, where the mean pixel intensity per cell was computed (n = 35–50 cells per treatment group). [...] All data is presented as mean ± standard deviation, unless otherwise noted. Nitrite release, gene expression of IL-1β and aquaporin, and cell size were analyzed with two-way ANOVA and Fisher LSD post-hoc test. Intracellular water content and hydraulic permeability were compared with repeated measures ANOVA and Fisher LSD post-hoc test with treatment and osmolarity step as variables. Linear regression analysis was performed between and cell radius in each treatment group. Regression coefficient (R) and p-statistics from the regression analysis are reported. All analysis were performed with STATISTICA software (StatSoft, Tulsa, OK, USA), with p<0.05 considered significant. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Bos taurus
Diseases Intervertebral Disc Degeneration