H2dcfda

ROS production levels were measured using 2′7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen, USA) in hBMSCs cells. The DAF-FM assay kit (Invitrogen, USA) was used as a total RNS indicator according to the manufacturer’s instructions. The cells were cultured in 6-well plates for cell fluorescence detection after preparation as described above (Corning, Midland, NC, USA). The cells were incubated with 100 M hydrogen peroxide (H₂O₂) for 30 min to generate a positive control group. The cells were incubated with 10 μM H₂DCFDA and DAF-FM probes for 30 min in the dark condition, and fluorescence intensity was observed under a confocal laser scanning microscope (Olympus, Hamburg, Germany). To further confirm our results, we prepared samples at the same procedure and then incubated them for 6 h to detect the intracellular ROS. Subsequently, each group of cells was collected for detection on flow cytometry.
The most substantial effects on stem cell differentiation were caused by H₂O₂ and nitric oxide (NO). We measured the extracellular H₂O₂ and NO levels in serum-free culture media following NBP treatment using the QuantiChrom TM Peroxide Assay Kit and the Nitric Oxide Assay Kit (Bio Assay System, CA, USA), respectively. Experimental procedures were performed in accordance with the manufacturer’s instructions.

The measurement of intracellular ROS levels was made using dihydrodichlorofluorescein diacetate (DCF-DA) in which the fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA; Molecular Probes Inc., Eugene, OR, USA), was converted by intracellular esterase to H₂DCF, which was oxidized by intracellular ROS to the highly fluorescent DCF. The OSSCE or MC treatment was administered for 10 min, and the cells were then stimulated with HG for 96 h. The cells were washed with Hank’s Balanced Salt Solution (HBSS) buffer and incubated in the dark for 30 min in HBSS buffer containing 50 µM H₂DCF-DA. The DCF fluorescence was measured using a Synergy HT spectrofluorometer (excitation 485 nm/emission 530 nm, BIO-TEK, VT, USA). The production of intracellular ROS was visualized by the fluorescence microscopic imaging of cells incubated in the dark for 5 min in a HBSS buffer containing 10 µM H₂DCF-DA, using an Olympus microscope (BX51, Olympus, Japan) equipped with an Olympus DP 70 camera.

We used 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Molecular Probes, Eugene, OR, USA) to measure the inhibition of PM_2.5-induced ROS by 3-BDB. Cells (1.0 × 10⁵ cells/mL) were seeded into a 6-well plate. Cells were added to 10, 20, and 30 μΜ of 3-BDB or 1 mM of N-acetyl cysteine (NAC) for 1 h and then exposed to 50 μg/mL of PM_2.5 for 30 min. Cells were stained with H₂DCFDA (25 μΜ), and stained cells were detected using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). Similarly, cells were seeded into the chamber slides, and 30 μΜ of 3-BDB were treated for 1 h and then treated with PM_2.5 (50 μg/mL) for 30 min. Cells stained with H₂DCFDA were observed using an FV1200 laser scanning confocal microscope (Olympus, Tokyo, Japan).

Intra-oocyte ROS level was measured by staining with 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCF-DA; Molecular Probes Inc., Eugene, OR, USA), adopted from Park et al. [33 (link)]. Intra-oocyte GSH content was measured with monochlorobimane (MCB), adopted from Keelan et al. [34 (link)]. Briefly, oocytes were denuded and incubated for 15 min either with 10 μM H₂DCF-DA or 12.5 μM MCB in 0.4% BSA-PBS at 38.5°C. Oocytes were washed in 0.1% BSA-PBS, immediately transferred with a 10-μL drop to a slide and observed under Olympus BX50 epi-fluorescent microscope with 10X magnification (H₂DCF-DA: λ_ex 460 nm, λ_em 525 nm; MCB: λ_ex 370 nm, λ_em 461 nm). Average fluorescence intensity per oocyte was measured with Image J software and normalized to the background average intensity.

The production of ROS was monitored using the ROS-sensitive fluorescent probe H₂DCFDA (Invitrogen, Waltham, MA, USA), as described by Wang et al. [15 (link)]. The epidermis was pre-treated with opening buffer for 1 h under light, then overlaid with 10 μM H₂DCFDA in the same buffer for 20 min in darkness. Thereafter, peels were superfused with the measuring buffer to remove excess dye. The peels were incubated in measuring buffer with and without 100 μM ABA for 30 min, and the H₂DCFDA fluorescence was monitored every 15 min using an Olympus FV3000 confocal microscope (Olympus, Tokyo, Japan). The H₂DCFDA was excited by the 488 nm line, and the fluorescence emission was collected through a 505 to 550 nm bandpass filter. The background fluorescence recorded prior to H₂DCFDA loading was corrected.

Then 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Oakville, Ontario, Canad) was used to stain nuclei, and a fluorescein diacetate (FDA; Sigma-Aldrich, Oakville, Ontario, Canad) assay was performed to assess the vitality of fresh pollen grains. Samples were washed, embedded, and stained as described previously [2 (link)].
For ROS detection, fresh pollen grains were washed with PBS, then incubated in 5 µM H₂DCF-DA (Sigma-Aldrich) dissolved in anhydrous dimethyl sulfoxide (DMSO; Sigma-Aldrich) for 60 min in the dark at 25 °C. Excess probe was removed using PBS before detection.
Fluorescent signals were captured using a fluorescence microscope (Olympus BX 51, Olympus, Japan). Filter sets for blue (DAPI, 4′,6-diamidino-2-phenylindole) and green fluorescence (FDA, H₂DCF-DA) were Olympus part numbers.

To detect intercellular ROS in living embryos, we used H2DCF-DA from Sigma-Aldrich (St. Louis, MO, USA). H2DCF-DA was prepared in DMSO immediately prior to loading. Embryos were incubated with 10μM H2DCF-DA for 10 minutes and observed under afluorescence microscope (Olympus, Japan), with an excitation wavelength of 480 nm and an emission wavelength of 505-530 nm.