Effect of CO donor on binding and dissociation of the LDV-FITC probe on resting and activated cells. LDV-FITC binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant FPR ΔST plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by addition of excess unlabeled competitor (LDV), and therefore, no autofluorescence can be seen. A. The experiment involved sequential additions of the LDV-FITC, and different concentrations of CORM-2 or vehicle. The non-specific binding of the probe was determined using LDV. Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves to the single exponential equation. B. The span of the single exponential fits for the dissociation curves (from A after LDV addition) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The sigmoidal dose-response (Hill slope = 1) was fit using GraphPad Prism. C. The sequential addition of the LDV-FITC, the high affinity FPR ligand (fMLFF), CORM-2 or vehicle, and LDV. LDV-FITC koffs were determined as described for A. The level of LDV-FITC binding corresponding to resting cells is indicated by the dashed line. D. The span of single exponential fits for the curves (from panel C) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The dose-response was fit analogously to B. E. The experiment involved addition of the LDV-FITC, RuCl3 or vehicle. F. The sequential addition of the LDV-FITC, and CORM-2. The "old" CORM-2 was prepared by incubating the solution for 48 hours at room temperature. The non-specific binding of the LDV-FITC probe was determined using LDV. For panels A, C, E, and F, a representative experiment of two independent experiments is shown.