Effect of endocytosis inhibitors on fMSC migration, attachment, chemotaxis and differentiation. A and B) Scratch wound assay was carried out in a 96 well plate using the Incucyte live cell imaging system. A confluent monolayer of fMSC was wounded and treated for 1 hr with serum-free treatment media, or treatment media with DMSO vehicle, 80 μM blebbistatin or dynasore. Images were taken 4 hourly, with representative images of media and blebbistatin treated cells at 0, 6 and 12 hr intervals shown in A. The grey overlay is the automatically generated wound outline at 0 hr. B) Quantitative analysis of percentage wound confluence (N = 3 donors, replicates of 5) show no statistically significant difference in ability to migrate into the wound zone for any cell treatment. C) fMSC were prestained with CFSE, then treated with either blebbistatin or dynasore before being placed into fibronectin or collagen I coated wells of a 96 well plate. Cell adhesion was determined by fluorescence intensity after a 1 hr incubation and removal of non-adherent cells. D) Untreated or inhibitor-treated cells were placed in the upper well of a Transwell plate. Cells were incubated for 4 hr at 37°C and migration determined at a range of concentrations of SDF-1 by fluorescence intensity in the bottom well was higher (p < 0.01** for both) in inhibitor-treated cells. fMSC were treated with either vehicle, 80 μM of blebbistatin or dynasore for 2 hours and the media then replaced with either (E) osteogenic- or (F) adipogenic-induction media. After 3 weeks culture, differentiation was determined by Alizarin Red (osteogenesis) or Oil Red O (adipogenesis) staining. Undifferentiated fMSC cultured in normal growth media are shown on the right.