Microclimate controlled stage top incubator

For tracking deformation in polyacrylamide (PAA) gels (Polio and Smith, 2014 (link); Ray et al., 2017 (link)) during traction force microscopy (TFM) analysis, we modified patterned PAA platforms by adding well-ultrasonicated 0.2mm fluorescent nanobeads (Polysciences) into PAA solutions (1:1000 dilution) before gel polymerization. ‘‘Before’’ and ‘‘after’’ cell removal images of the PAA micropatterns were taken with live confocal laser scanning at the interface planes between cells and the adhesion ligands patterns. Cell removal was performed by adding SDS detergent (Fisher Bioreagents, USA) to the final concentration of 0.5% (w/vol). Live cell imaging was performed in a microclimate-controlled stage top incubator (Tokai Hit, Japan) at 37° C in 5% CO₂. Bead displacements and corresponding traction forces fields were calculated using an iterative particle image velocimetry (PIV) algorithm and an unconstrained Fourier transform traction cytometry algorithm, respectively (ImageJ plugins)(Tseng et al., 2012 (link)).

For tracking deformation in PAA gels^{11 (link)} during TFM analysis, we modified patterned PAA platforms by adding 0.2 µm fluorescent nanobeads (Polysciences, Cat#BLI832-1) into PAA solutions before gel polymerization. Before and after removal of cells, images of the PAA micropatterns were taken with live confocal laser scanning at the interface planes between cells and the adhesion ligand patterns. Live cell imaging was performed in a microclimate-controlled stage top incubator (Tokai Hit, Japan) at 37 °C in 5% CO₂. Bead displacements and corresponding traction forces fields were calculated using an iterative particle image velocimetry algorithm and an unconstrained Fourier transform traction cytometry algorithm, respectively (ImageJ plugins).