FIG. 4.

Control experiments. (A) pH measurements in PBS-I before and after addition of 1 to 5 mM H₂O₂ (n = 3). (B) and (C) Single-cell chronoamperometric experiments (at 650 mV) with a 10 μm Pt-UME versus Ag/AgCl in PBS-I with the electrode fixed 5–8 μm above the MC. (B) Addition of SOD (50 U/mL) to a TPA-stimulated MC (example for n = 3). (C, D) TPA stimulation of MCs pretreated either with the pan NOX inhibitor VAS2870 (20 μM in DMSO; 30 min; red trace) or with DMSO (black trace; examples for n = 5 per condition). (E, F) Comparison of normalized ESR spectra from experiments with MC suspensions (depicted in Fig. 7D, E) to test for NO^● formation by TPA-stimulated MCs. Experimental conditions and settings of the ESR spectrometer are detailed in the Materials and Methods section. Black traces are from DMSO treated (control; n = 3) and red traces from cells about 35 min after TPA application (n = 3). Spectra in (E) and the enlarged cut out in (F), corresponding to the magnetic field interval marked by the red bar in (E), show complete overlay of the normalized spectra. Because of the low intensity, the signal-to-noise ratio of the spectra from unstimulated MCs is higher than from stimulated cells. ESR, electron spin resonance; SOD, superoxide dismutase.