Oris plate

To assess cell motility, 120,000 GM-CSF/IL3-derived BMMO were seeded in complete culture medium into wells of the 96-well ORIS plate (Platypus Technologies, WI, USA) containing a round silicon insert set in each well. Cells were incubated overnight to settle before plugs were removed with the supplied tool. Media was carefully aspirated and replaced with fresh media. Each well was inspected using the microscope, noting wells with disturbed exclusion zones: these wells were not used. Images of wells were captured at this time to measure original wound size and used as timepoint 0. Cells were either left unstimulated, stimulated with 25 μM clozapine, or 0.001 μM Latrunculin A (Sigma-Aldrich) as a positive control or the respective vehicles. Plates were incubated for 3 days before images of each well were taken to measure final wound size. Cell viability was assessed via MTT assay at the end of the experiment.
ImageJ running the MRI Wound Healing Tool macro (Montpellier RIO Imaging, Montpellier, France) was used to measure wound size from images using the following script parameters: method: variance; variance filter radius: 5; threshold: 50; radius open: 1; min. size: 10,000. Compound-induced changes to wound closure were assessed using the following equations:
$$\text{Closure}\left(\%\right)=\left(\frac{{\text{wound area}}_{\text{day}0}-{\text{wound area}}_{\text{day}3}}{{\text{wound area}}_{\text{day}0}}\right)\times 100$$ $$\text{Closure},\mathrm{as}\%\text{vehicle}=\left(\frac{\text{closure}{\left(\%\right)}_{\text{test compund}}}{\text{closure}{\left(\%\right)}_{\text{vehicle}}}\right)\times 100$$

hUCB-MSCs (3 × 10² cells)/100 μl were seeded in each well of Oris plate (Platypus Technologies, Madison, WI, USA) and incubated for at least 24 h to permit cell adhesion. The cells were cultured until they reach around 70% confluence. After that, inserts were carefully removed and the cells were gently washed with warm PBS. The cells were incubated with 10 μM of AA in serum-free medium for 24 h and then treated with 5 μM of calcein AM for 30 min to stain the cell populations in endpoint assays. By using a microplate reader to measure excitation/emission wavelengths (485/515 nm), migrated cells were quantified.

Peritoneal macrophage migration assays were described previously [46 (link)]. Cells were seeded in each well of Oris plate (Platypus Technologies, Madison, WI, USA). The inserted column were removed carefully after 24h until the seeded cells attached to the plate. Inserts were then carefully removed and cells gently washed with warm PBS. The cells were incubated with or without LPS (100 ng/ml) in RPMI medium for 24 h and then stained with 5 μM of calcein AM for 30 min. BMG Galaxy fluorescent microplate reader was used to measure excitation/emission wavelengths (485/515 nm) of migrated cells into the previously restricted zone.