H2dcfda

Bone marrow neutrophils were collected from Ncf1^fl/fl and Ncf1^Lyz–/– mice using the Mouse Neutrophil Isolation Kit (Miltenyi Biotec) based on the manufacturer’s instructions. Neutrophils were resuspended in prewarmed medium (RPMI-1640 containing 10% FBS and penicillin-streptomycin) containing the probe H2DCFDA (Thermo Fisher) at the working concentration of 20 μM and incubated for 30 minutes at 37°C. Then 100 ng/ml PMA or 100 ng/ml LPS was added into the medium and incubated for an additional 30 minutes at 37°C. H2DCFDA intensity was measured by flow cytometry (CytoFLEX, Beckman Coulter) in the FL1 channel.

For the measurements of intracellular ROS level, fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen, D-399) was used. H2DCFDA was dissolved in DMSO to obtain a 10 mM stock solution which was further diluted in PBS before use to obtain staining solution. Cells were incubated with 5 μM H2DCFDA staining solution in the dark for 20 min at 37°C, then harvested with 0.05% trypsin-EDTA solution, suspended in a fresh medium and immediately analyzed with CytoFLEX flow cytometer (Beckman Coulter, USA; 488 nm laser). Cells were detected by size and granularity using FSC/SSC dot plot, and cell debris was gated out. Mean fluorescence intensity from 10,000 cells was acquired.

We used the ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate to determine the intracellular ROS levels (H2DCFDA, SIGMA-ALDRICH Corp., St. Louis, MO USA). Following intermittent hypoxia/reoxygenation (IHR) stimulation, cells were incubated in the dark for 30 min with 5 M H2DCFDA and immediately analyzed using a flow cytometer set to 488 and 535 nm excitation and emission wavelengths, respectively (Beckman Cytomics™ FC500, Beckman Coulter, Brea, CA, USA).

The production of reactive oxygen species (ROS) was measured using the ROS-sensitive dye carboxy-2, 7-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen) as an indicator. Briefly, glioma cells were homogenized in assay buffer, and the resulting homogenates were incubated with H2DCFDA at 37°C for 3 h. The fluorescent product formed was quantified using a spectrofluorometer that was set at 485/525 nm (Beckman, USA), and changes in fluorescence were expressed in arbitrary units.

The ROS measurement protocol was adapted from Hoeksema, M. et al.’s study [60 (link)]. HPF (ThermoFisher Scientific, Waltham, MA, USA) and H2DCFDA (ThermoFisher Scientific), of which the fluorescence signal was proportional to the ROS, were used. The bacterial culture was diluted 1: 500 in LB medium and grown until OD₅₉₅ of 0.6. Stationary phase bacteria were prepared by harvesting the overnight culture and washing and diluting it to 1:10 in M9 minimal medium. The fluorescent dye, at a final concentration of 5 µM, was added to the bacterial suspension and added to a 96-well black opaque plate (SPL) in a volume of 200 µL per well. Bacterial cells in different wells of the 96-well plates were treated with different antibiotic(s) and/or compounds, and the untreated control was included for comparison. The antibiotic treatments were performed in triplicate. HPF or H2DCFDA fluorescence (485 nm excitation, 535 nm emission) was measured in a DTX 880 Multimode Detector (Beckman Coulter, Brea, CA, USA) or SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) in a kinetic program consisting of 16 cycles of measurement at 30 min intervals at 37 °C, with shaking. OD₅₉₅ of the bacterial culture was also recorded for normalization. Experiments related to H₂O₂ were included in Supplementary Methods.