Media were changed every 3 days, and explants were cultured for a total of 14 days at 37 C 5% CO2. Cell proliferation NSC 125973 analyses in meniscal repair explants On Day 14, explants were transected vertically to allow visualization of cells throughout the cross section. Explants were labeled using a modified protocol based on the Click iT EdU Alexa Fluor Inhibitors,Modulators,Libraries 488 Imaging Kit. Briefly, explants were fixed with 3. 8% formaldehyde for 30 minutes, permeabilized with 0. 5% Triton X 100 for 30 minutes, and tagged with the Alexa Fluor dye to label all proliferated cells. To stain all cells, explants were washed in TE, pH 7. 4, stained for 30 minutes in the dark with 1 uM Syto 82 nucleic acid stain, and then washed three times with TE. Cells were visualized and photographed using a confo cal laser scanning microscope as described above for the micro wounding assay.
Images were collected more than 50 um into the face of the sample to ensure that cells damaged during transection were excluded. Two images per location were collected from the outer Inhibitors,Modulators,Libraries ring, inner core and repair interface for both the surface and cross section of the explants. Meniscal repair model explant image analysis Data were collected from different areas of the surface and cross section of the meniscal repair model explants. Measured areas were outlined in Zeiss LSM image browser. The surface interface included a 50 um region on either side of the interface, inclusive of the interface, at the surface of the tissue. The surface region of the tissue included images from both the inner Inhibitors,Modulators,Libraries core and outer ring that were outside this defined interface region.
For cross section images, the tissue was divided into three distinct layers based on distance from the surface and cell morphology. The first 50 um from the surface was defined as the superficial zone, the next 100 um was defined as the middle zone, and the next 300 um was termed the deep zone. The cross section interface included a 50 um region on either side of the interface, Inhibitors,Modulators,Libraries inclusive of the interface, for each of these three layers. The cross section of the tissue included images of the cross section from both the inner core and outer ring that were outside the defined interface region. Confocal images were exported as separate green and red channel images and saved as TIFF files.
The Inhibitors,Modulators,Libraries images were gray scaled using Adobe Photoshop and processed using Scion Image to invert, subtract background by removing 2D streaks, and smooth. Optimal threshold values were determined for the green and red channels individually. Proliferated cells and total cell counts were obtained using intensity thresholds of 75 and 40, respectively, and a minimum particle size of five. Cell Pacritinib mw counts were obtained for each of the defined regions in the surface and cross sectional planes.