X71 fluorescent invert microscope

MSCs (2.4×10³ cells/mm²) were cultured onto the basal surface (lower chamber) of 0.4 or 3 µm pore 6-well Transwell inserts for 24 h as described previously (Munir et al., 2016 (link), 2017 (link); McGettrick et al., 2017 (link)). The culture medium was removed from both chambers. Phosphate-buffered saline with Ca²⁺ and Mg²⁺ (PBS; Sigma) was added to the lower chamber. CD41a-labelled whole blood was added to the apical surface (upper chamber) of the filter for 1 h, before non-adherent cells were removed by washing. Adherent platelets were imaged using an Olympus X71 Fluorescent Invert microscope enclosed at 37°C. Adhesion and platelet aggregation were quantified by using the Particle Analysis of Fluorescence function in ImageJ (NIH) and expressed as platelet coverage in µm².

Rat-tail collagen type 1 (2.15 mg/ml; First Link Ltd, West Midlands, UK) was mixed with 10× M199 (Gibco, Thermo-Fisher), and then neutralised by addition of 1 N NaOH on ice, as described (Jeffery et al., 2013 (link); Munir et al., 2017 (link)). The gel was allowed to set for 15 min at 37°C, and then equilibrated for 24 h with culture medium. MSC spheroids were formed by suspending 2.5×10⁴ cells in 35 µl culture medium as a hanging droplet for 48 h, before settling onto the surface of the collagen gel. MSC migration over the collagen gel was assessed at 24 h and 48 h using an Olympus X71 Fluorescent Invert microscope enclosed at 37°C. Images were analysed off-line using AngioSys2.0 software (Cellworks, Buckingham, UK) to quantify the number of marginal cells (those cells disseminating from the edge of the spheroid on the surface of the collagen gel). Data were expressed as the number of marginal cells migrating away from the spheroid and along the surface of the collagen matrix as a percentage of the total number of cells seeded.