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Fig. 1 | Biological Procedures Online

Fig. 1

From: DNA Area and NETosis Analysis (DANA): a High-Throughput Method to Quantify Neutrophil Extracellular Traps in Fluorescent Microscope Images

Fig. 1

DANA operates in two parts to provide information on NETosis and cellular DNA area. In DANA_I, images are loaded into Fiji where they are thresholded, analyzed, and information on each region of interest (ROI) is outputted to a .csv file for each image of the sample. Once all images for a single sample have been processed within DANA_I, DANA_II then loads the ROIs from all .csv files from that sample. ROIs with raw integrated densities smaller than the lower cutoff value (LCV) or larger than the mean plus the product of the upper elimination cutoff parameter (UECP) and the sample standard deviation are excluded to eliminate fragments and multiples. Of the remaining ROIs, the average area of the five smallest ROIs is computed (denoted as x-bar). The five smallest non-excluded ROIs are considered the most condensed nuclei. The areas of all remaining ROIs are divided by x-bar (or by a user defined value if required by future users) and ROIs are labelled as NETs if their relative area is greater than the NET cutoff. Data on NETs and DNA area is exported to an image-specific .csv file. These image-specific files also contain information on the percent NETosis and average cellular DNA area for that specific image. Additionally, a summary.csv file is exported with information on the percent NETosis and average cellular DNA area for the entire sample. Outlines in the rightmost circle indicate ROIs. The arrowhead in the rightmost circle indicates multiple cells present within a single ROI

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