Inhibition of mTOR impairs FPR-induced PMN chemotaxis and blocks mitochondrial activity. (A and B) Freshly isolated primary human PMNs were exposed to a chemotactic gradient using 100 nM fMLP in a micropipette, and cell migration was monitored. The chemotactic behavior of untreated cells and of cells pretreated for 30 min with 1 µM rapamycin or 1 µM PP242 was recorded (see also Video 5, top). Data shown are representative of results obtained with cells from at least three different healthy individuals. (B) Migration speed, effective migration speed, and correct direction were calculated as described for Fig. 1. Data shown are expressed as mean ± SD, and accumulated results are from using cells from at least three different individuals; n = 20–40 cells in each experiment. Statistical analysis was done with one-way ANOVA; *, P < 0.05. (C and D) Mitochondrial Ca²⁺ uptake in PMNs was assessed with 1 µM Rhod2 as described in Fig. 2 by using a fluorescence microscope (DMI6000 B; Leica; objective: 100× oil, NA 1.30; DFC365 FX camera; Leica). Cells were stimulated with 1 nM fMLP in the absence or presence of 1 µM rapamycin or 1 µM PP242. Data shown are means ± SD (error bars) of normalized gray values of 15–25 cells (bar, 10 µm; see also Video 6).