Journal: Frontiers in Pharmacology

Article Title: An Automated Quantification Tool for Angiogenic Sprouting From Endothelial Spheroids

doi: 10.3389/fphar.2022.883083

Figure Lengend Snippet: Schematic demonstrating setup of spheroid assay for angiogenesis sprouting. (A) Human umbilical vein endothelial cells (HUVEC) are fluorescently dyed with CellTracker Green CMFDA (5 μm) and seeded as hanging drops for 24 h. Spheroids are collected, resuspended in a fibrinogen/methylcellulose solution, and mixed with thrombin in a 96-well plate to embed spheroids within the generated fibrin gel. Medium containing pro- or anti-angiogenic factors is added 1 h later to initiate sprouting. Angiogenic sprouting is imaged 24 h later using a real-time, fluorescence microscopy plate reader. (B) Representative images of embedded spheroids in a single well and a close-up of sprouting from a single spheroid. Images were acquired using phase contrast and fluorescence microscopy. Scale bar of whole-well image = 2000 μm.

Article Snippet: We subsequently developed a Matlab-based automated algorithm to quantify angiogenic sprouting from HUVEC spheroids imaged using fluorescence microscopy ( ).

Techniques: Generated, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: An Automated Quantification Tool for Angiogenic Sprouting From Endothelial Spheroids

doi: 10.3389/fphar.2022.883083

Figure Lengend Snippet: Quantification method of angiogenic sprouting from HUVEC spheroids. (A) Images demonstrating the automatic generation of masks and skeletons used to quantify total area, sprout area, and cumulative sprout length from each spheroid. The original image (1) is intensity adjusted using adaptive histogram equalization (2), and segmented using a combination of Sobel segmentation, convolution, and adaptive thresholding to generate a spheroid mask (3), as described before . The image was then reduced to an initial skeleton using a skeletonization method (4). After the spheroid center (5) and sprout area (6) were identified, the center was subtracted from the sprout skeleton to exclude extensions found in the spheroid center (7). Small extensions were then removed to generate the final sprout skeleton (8). (B) Images demonstrating the counting of individual sprouts as migrated or attached. Each sprout in the skeleton is assessed as to whether it intersects with the skeleton of the center. If a sprout intersects with the center, it is considered “attached”; otherwise, it is considered “migrated”. (C) View of output csv file from automated algorithm.

Article Snippet: We subsequently developed a Matlab-based automated algorithm to quantify angiogenic sprouting from HUVEC spheroids imaged using fluorescence microscopy ( ).

Techniques: