dHL-60 cells expressing a previously characterized CDC42-FRET sensor (19 (link), 23 (link)) were resuspended in warmed modified L-15 media containing 2% FBS at a density of 500,000 cells/ml. To avoid washout, cells were plated on 96-well optical-glass bottomed imaging plates (Cellvis, catalog # P96–1.5H-N) coated with poly-D-lysine (“PDL”, Sigma; catalog # P6407). In a sterile tissue-culture hood, 50 μl of 200 mg/ml PDL was added to each well and incubated at room temperature for 30 minutes. After incubation, wells were washed twice with 50 μl sterile DPBS (Life Technologies; catalog # 14190250) and dried thoroughly at 65°C for at least 30 minutes before 100 μl of cells were added (results in ~40–50k cells/well). Before imaging, plated cells were incubated at 37°C for 30 minutes to allow adherence. Time-lapse microscopy was performed using a Nikon Ti-E inverted microscope with a 20x (0.75 NA) Plan Apochromat objective. Images were taken at 5 second intervals. A flash of red light immediately preceding the 4th frame was used to signal the timing for the addition of 100 μl of chemoattractant or media by pipet. To achieve rapid mixing, chemoattractants were added to media at a 1:1 ratio. Images were captured using 2×2 binning. For FRET experiments, Dual Zyla-4.2-USB3 sCMOS cameras were used to capture CFP and YFP channels simultaneously.