Next, we established a model system for evaluating our probe in cells. HEK293T cells were transiently transfected with C-terminally FLAG-tagged FPR1, yielding a diffuse band by Western blot that resolved to give a tighter band of the expected molecular weight upon deglycosylation with PNGaseF (Supp. Figure ). Flow cytometry analysis of HEK293T cells incubated with 10 nM Tracer-FITC showed a clear shift to a population of fluorescent cells with minimal background binding in mock-transfected cells (Figure ). We were pleased to observe that probe-TAMRA gave a similar increase in fluorescence in flow cytometry analysis at the relevant wavelength (Figure ) and titration of probe from 0-200 nM showed low background binding that did not exceed 15% of total binding (Figure ). Following background subtraction, a KD of 3.5 ± 0.9 nM can be calculated for Probe-TAMRA. The different fluorophores in the tracer and probe should allow both to be monitored during competition experiments. Incubation of FPR1-expressing cells with Tracer-FITC or Probe-TAMRA showed clear populations of cells at the appropriate wavelengths with little crosstalk between the fluorophores (blue and cyan dots in Figure ). Co-incubation of the tracer and probe showed clear competition (red dots, Figure ). We then performed a titration, keeping the concentration of Tracer-FITC constant at 10 nM and increasing the concentration of probe-TAMRA up to 200 nM. This gave the expected decrease in FITC signal with a concomitant increase in TAMRA signal (Supp. Fig. ) and a plot of the percentage of maximum fluorescence for each showed concentrationdependent competition (Figure ). These data indicate that the probe and tracer most likely bind the same site. We then attempted competition experiments with known ligands. We could only observe competition of probe-TAMRA binding with the canonical FPR1 ligand fMLF at concentrations greater than 5 µM (Supp. Figure ), which is surprising given that the KD of this tripeptide is reported in the nM range. However, a survey of the literature showed that fMLF is often used at micromolar concentrations to achieve competition and that this is highly dependent on the competing fluorescent ligand. Pleasingly, known antagonist Boc-MLF could outcompete probe-TAMRA at concentrations of 10-50 µM, consistent with the literature (Supp. Fig. ). Together these data suggest highly specific binding of probe-TAMRA to FPR1.