3/11/2024 0 Comments Nih imagej fijiInstantaneous speeds for leader mobile (LM) cells transfected as in ( A). Instantaneous speeds for all cells transfected as in ( A). Percent of no leader (NL), leader non-mobile (LNM), and leader mobile (LM) for melanoma A375-M2 cells stained with membrane dye (n = 124 cells), transiently transfected with EGFP alone (n = 92 cells), EGFP-F-tractin (n = 43 cells), mEmerald alone (n = 37 cells), or mEmerald-LifeAct (n = 47 cells). All data are representative of at least three independent experiments. Statistical significance was determined using a One-way ANOVA and a Tukey’s post hoc test. Dashed red outline highlights average whole cell roundness and average whole cell aspect ratio as shape descriptors that can differentiate between leader non-mobile (LNM) and no leader (NL) cells. Shape descriptors for the whole cell across each phenotype. Dashed red outline highlights the average cell body size as a shape descriptor that can differentiate between leader non-mobile (LNM) from no leader (NL) cells. Shape descriptors for the cell body across each phenotype. Solid red outline highlights the average largest bleb size as a shape descriptor that can differentiate between leader mobile (LM) from leader non-mobile (LNM) cells. Shape descriptors for the largest bleb across each phenotype. Average number of blebs ( left) and bleb size ( right) for each phenotype. Instantaneous speeds ( left) and top instantaneous speeds ( right) for each phenotype. Furthermore, that F-tractin increases cell stiffness, which was found to correlate with a decrease in migration, thus reaffirming the importance of cell mechanics as a determinant of Leader Bleb-Based Migration (LBBM).Ī. We also demonstrate, using flow cytometry, that live markers increase total levels of F-actin. As validation, we show that Analyze_Blebs can detect significant differences in cell migration and morphometrics, such as the largest bleb size, upon introducing different live markers of F-actin, including F-tractin and LifeAct tagged with green and red fluorescent proteins. Here, we demonstrate that a novel Fiji/ImageJ-based plugin, Analyze_Blebs, can be used to quickly obtain cell migration parameters and morphometrics from time lapse images. However, as this is a nascent area of research, few tools are available for the rapid analysis of cell behavior. When confined, cells have recently been shown to undergo a phenotypic switch to what has been termed, fast amoeboid (leader bleb-based) migration.
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