Quantifying Matrix Degradation by Cells

Coverslips coated with Oregon Green 488-conjugated gelatin (Life technologies, ThermoFisher) were prepared as described in Thuault et al. [30 (link)]. After seeding for 4 h on gelatin-coated coverslips rehydrated in complete growth medium for 1 h before use, cells were fixed with a solution of 4% formaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA. When EB1 was labeled, cells were fixed with methanol before a 2nd fixation step with 4% formaldehyde in PBS and blocking with 1% BSA. Antibodies directed against the target proteins and secondary antibodies labeled with DyLight 405 or AlexaFluor 594 (Jackson ImmunoResearch, West Grove, PA, US) were then used for immunolabeling. A Zeiss structured light ApoTome microscope equipped with a 63× 1.4 plan ApoChromat objective was used for image acquisition (Zeiss, Jena, Germany). Ten random fields per coverslips, using 2 coverslips per condition per experiment, were imaged to assess the percentage of cells degrading. A home-made Fiji macro was used to analyze matrix degradation. The mean degraded area and the number of degradation foci per cell were quantified for 25 cells per condition per experiment.