Computational protocol: Reduced cell cohesiveness of outgrowths from eccrine sweat glands delays wound closure in elderly skin

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Protocol publication

[…] Area of new epidermis was measured on calibrated still, top view 3D images with reconstruct 1.1. Percent re‐epithelialization = epidermis area/total biopsy area × 100. Epidermal thickness was not taken into account in these calculations. Epidermal volumes were extracted from calibrated traces in reconstruct.Appendage (i.e., PSU and ESG) count was made on still or animated 3D renderings. Percent contributing ESGs to wound closure = number of ESGs topped with an outgrowth/number of total ESGs × 100. Similar equation was used for PSUs. For each sample, all outgrowths that appeared shared between an ESG and a PSU (i.e., was of uncertain origin) were not included in calculation. When several biopsies were available for an individual (e.g., multiple time points), results were grouped.Ki67 staining density was calculated from raw data generated in reconstruct, dividing the Ki67 count by the total volume of the respective appendage subunit (ESG ducts and coils, hair follicles, sebaceous glands, and arrector pili muscles). For ESGs, ‘ducts’ designates the descending portion of the gland until the first elbow. Each average was calculated from subunits of 11 young and 14 aged ESGs, and 9 young and 5 aged PSUs from 2 to 3 subjects per age group, except for aged arrector pili (n = 3).Laminin‐γ2 IHC staining density was measured with imagej 1.49v (http://imagej.nih.gov) on the isolated red staining channel, using percent area from pixel counts of the thresholded image. Staining outside the dermal–epidermal area, if any (e.g., around appendages) was excluded. Four‐to‐six 400× images encompassing the width of the biopsy were analyzed for each subject. Measurements were averaged among three subjects per age group.Extent of cell separation (‘area of cell gaps’) was measured with imagej and represents areas of background color within the epidermis. Pixel counts were normalized to the total surface of the epidermis (area between dermal–epidermal junction and top of granular layer (stratum corneum excluded)). Three images each of random but not overlapping location across the width of the biopsy were analyzed for each subject. Measurements were averaged among three subjects per age group.Surface area occupied by individual cells was measured with IMAGEJ on β1‐integrin IHC‐calibrated digital images (400× magnification). A minimum of 10 basal keratinocytes was evaluated for each image, and measurements were averaged among 6 subjects per age group.Number of basal keratinocytes was evaluated on β1‐integrin or laminin‐γ2 IHC‐calibrated digital images with imagej (cell counter plugin, http://imagej.nih.gov/ij/plugins/cell-counter.html) and normalized to length of dermal–epidermal junction (also measured with imagej). The entire width of 2 randomly taken 400× images was used for each subject, and measurements were averaged among 6 subjects per age group.Epidermal thickness was evaluated on histology or IHC‐calibrated digital images with imagej: length between dermal–epidermal junction and top of granular layer (stratum corneum excluded) was measured roughly every 100–150 μm, on the entire width of 2–4 randomly chosen 100× images for each subject, for an average of 35 measurements per time point. Values for each time points were averaged among 7–9 subjects per age group. [...] Apoptosis was detected via DNA fragmentation by the TUNEL assay with the ‘ApopTag® Red In Situ Apoptosis Detection’ kit (EMD Millipore) following the manufacturer's instructions. Positive control was obtained by digesting unwounded skin samples with 2.5 U DNase I (Sigma, Saint‐Louis, MO, USA) for 10 min at room temperature. Positive fluorescent staining was pseudocolored in red and DAPI nuclear counter staining in blue. Blue and red signals were separated from skin autofluorescence signal with the spectral unmixing plugin of imagej. […]

Pipeline specifications

Software tools ImageJ, Spectral Unmixing Plugins
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens