H2dcfda

The production of intracellular ROS (iROS) was measured by the cell-permeable fluorescence probe chloromethyl-29, 79-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Cat# C6827; Invitrogen) or 2′,7′-Dichlorofluorescin Diacetate (H₂DCFDA; Cat# 287810; Calbiochem) as described [27 (link)]. Briefly, mixed-glia cultures in black 96-well plates were pretreated with vehicle or G6PD inhibitors for 30 min followed by treatment with LPS for 6 h in phenol red-free medium. After incubated with 10 μM H₂DCFDA for 1 h at 37 °C, the fluorescence density was read at 488 nm for excitation and 525 nm for emission using a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices). Alternatively, microglia-enriched cultures grown in glass chambers were transfected with G6PD siRNAs or scramble siRNA for 30 h or pretreated with vehicle/G6PD inhibitors for 30 min followed by treatment for 18 or 24 h with LPS as described in the figure legend. The cultures were stained with 10 μM CM-H₂DCFDA or antibody against 4-hydroxynonenal (4-HNE; 1:500; Abcam). Brain sections were immune-stained for 3-nitrotyrosine (3-NT; 1:500; Abcam). Fluorescent images were collected using Zeiss LSM 780 or 880 confocal microscope and analyzed by using ImageJ software.

BALF cells were also used to determine whether ROS production was induced by the wildfire PM exposure. We used nonfluorescent 2’ , 7’-dichloro-fluorescein diacetate (H₂DCFDA, Invitrogen, Carlsbad, CA) to measure the intracellular ROS production because H₂DCFDA easily diffuses into cells and is converted to highly fluorescent DCF by intracellular ROS. At 24 h post-exposure, BALF cells were assessed by plating 10,000 cells onto cell culture plates and incubating the cells with H₂DCFDA at 10 μM for 2 h. Fluorescence of the probe was quantified using a fluorescence plate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA), with excitation at 485 nm and emission at 530 nm.

L. plantarum ST-III was cultivated in TYC medium under AE or AN conditions, as described above. After 12 h cultivation, bacteria cells were harvested by centrifugation at 5,000 rpm for 2 min and the level of reactive oxygen species (ROS) was determined using 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA; Beyotime Institute of Biotechnology, Haimen, China) according to the instructions provided by the manufacturer. In brief, around 1 × 10⁶ cells were collected and washed with phosphate-buffered solution (0.01 M phosphate, pH 7.4, PBS) and then treated with 10 mM H₂DCF-DA dissolved in PBS at 37°C anaerobically for 20 min. After removal of H₂DCF-DA and three times wash with PBS, the fluorescence intensity was monitored with excitation wavelength at 488 nm and emission wavelength at 525 nm on SpectraMax M5, Molecular Devices (San Jose, CA, United States).
For each sample, an equal amount of cells were sonicated and subjected to quantification of the total protein using the Bradford method. The fluorescence intensity was normalized with the total protein content, and the relative amount of ROS is expressed as DCF fluorescence intensity per milligram total protein, as described previously (Yan et al., 2018b (link)). The experiments were performed in triplicate and the average values, as well as the standard deviations, are shown.

To detect intracellular ROS, the fluorescent reporter dye 3′-(p-hydroxyphenyl) fluorescein (HPF, Invitrogen), 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetylester (CM-H2DCFDA, Life Technologies) and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen) were used, as previously described [20 (link)]. Briefly, 1 ml samples were collected after treatment and then resuspended in 1 ml of PBS containing 10 μM HPF, CM-H2DCFDA or H2DCFDA, respectively. Samples were incubated in dark for 20 min. The cells were then pelleted, the supernatant removed, and were resuspended in 1 ml filtere-sterilized PBS. Two hundred microliters of the resultant cell suspension were transferred to a dark 96-well plate. Fluorescence signals were measured using a SpectraMax M2 Plate Reader (Molecular Devices) with excitation/emission wavelengths of 490/515 nm (HPF), 495/520 nm (CM-H2DCFDA and H2DCFDA). The results shown represented the mean of one representative assay performed in triplicate, and error bars represent standard deviation. Statistical analysis was carried out with Student’s t-test.

The cells were incubated with 10 µM of H₂DCF-DA (Thermo Fisher Scientific) or 1 µM of Mito-SOX Red Mitochondrial Superoxide Indicator (Mito-SOX) (Thermo Fisher Scientific) for 30 min. Following this, the cells were exposed to methylmercury for the indicated condition. The cells were rinsed with PBS and the medium was changed to Hanks' balanced salt solution (HBSS). Fluorescence was detected by the plate reader, SpectraMax Gemini XPS (Molecular Devices, San Jose, CA, USA), set at Ex 490 nm/Em 530 nm (H₂DCF-DA) or Ex 510 nm/Em 580 nm (Mito-SOX).