Fig. 4

Diminished intracellular NADPH-oxidase derived ROS production by isolated eosinophils. Blood granulocytes from six healthy donors were isolated by Ficoll-Paque. Some granulocytes (Gran) were kept on ice and the rest was subjected to magnetic beads in order to isolate eosinophils (Eos) using magnetic beads. a Representative microscopic cytospin images, from one donor out of three, of granulocytes and purified eosinophils stained with May-Grünwald/Giemsa. b The histograms show the auto fluorescence of unstained eosinophils (black) as compared to unstained granulocytes (grey) in the FITC-channel as analysed by flow cytometry (n = 4). c-h ROS-production by granulocytes and isolated eosinophils from the same donor was measured on a Bioluminat LB9505 using (c-f) luminol-amplified CL to measure intracellular ROS, and (g-h) isoluminol-amplified CL to measure extracellular ROS. The left lane shows one representative trace of ROS production (Mcmp) over time (minutes; min), and the right lane shows the peak ROS-production (Mcpm) by isolated eosinophils (black line; black dots) or granulocytes (grey lines; grey dots) stimulated with (c-d) ionomycin (0.5 μM) and (e-h) PMA (50 nM). Statistical analysis in d, f and h was performed using a paired Student’s t-test after subtracting the stimulus induced peak-value to the value at time 0 min