Total ROS production was evaluated in PMNs by two methods. Cells were incubated (30 min) with the fluorescent probe (5 x 10−6 mol/L) 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) [24 (link)]. First, ROS production was assessed by fluorimetry using a Synergy Mx plate reader (BioTek Instruments, Winooski, VT). Second, it was assessed using a fluorescence microscope (IX81, Olympus, Hamburg, Germany) coupled with the static cytometry software ‘ScanR’ version 2.03.2 (Olympus). For static cytometry, PMNs from each subject were seeded in triplicate in 48-well plates and 16 images per well were recorded and analyzed [24 (link)].
The fluorescent probe Mitosox Red (5μM) was employed to assess mitochondrial ROS production. Leukocytes were seeded in 48-well plates and incubated for 30 min with the respective fluorochrome and washed with HBSS. 16 images per well were recorded with an IX81 Olympus fluorescence microscope (Olympus, Hamburg, Germany), and the static cytometry software ‘ScanR’ version 2.03.2 (Olympus) was used to quantify fluorescence individually (per cell). Fluorescent probes were purchased from Invitrogen (Life Technologies, Barcelona, Spain).
The fluorescent probe Mitosox Red (5μM) was employed to assess mitochondrial ROS production. Leukocytes were seeded in 48-well plates and incubated for 30 min with the respective fluorochrome and washed with HBSS. 16 images per well were recorded with an IX81 Olympus fluorescence microscope (Olympus, Hamburg, Germany), and the static cytometry software ‘ScanR’ version 2.03.2 (Olympus) was used to quantify fluorescence individually (per cell). Fluorescent probes were purchased from Invitrogen (Life Technologies, Barcelona, Spain).