To monitor changes in Cdc42 activity in live cells we used a previously characterized dimerization-optimized reporter for activation single chain Cdc42 biosensor (generous gift from Yi Wu, UConn Health, Farmington, CT, USA; Reinhard et al., 2017 (link)). Experiments were carried out as previously described for a similar Rac sensor (Baker et al., 2020 (link); Cooke et al., 2021 (link)). Briefly, SUM159 cells stably expressing low levels of the FRET sensor together with a marker for invadopodia (mCherry-cortactin), were treated with PDBu to induce invadopodia. We used 1:100 Oxyfluor reagent (Oxyrase Inc.) and 10 mM DL-lactate (Sigma-Aldrich) to reduce oxygen free radicals. Cells were imaged every 15 s with 2 × 2 binning and 16-bit depth, using a Nikon Eclipse Ti2 microscope equipped for FRET imaging (see Microscopes). Raw images were processed in batch using a custom designed macro in ImageJ, which included corrections to account for background and bleaching, and a median filter with a 1-pixel radius was applied to reduce noise artifacts. The FRET ratio was calculated, and a 16-color lookup table was applied to allow for the visualization of Cdc42 activation.