Figure 2

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 (k_off) 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 k_offs 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.