H2dcfda

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.

The levels of ROS were determined in bone marrow aspirate, spleen tissue lysate, blood and liver tissue lysate using 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Beyotime Institute of Biotechnology, Haimen, China). The oxidation of 2’-7’ dichlorofluorescin (H2DCF) to 2’-7’dichlorofluorescein (DCF) has been used quite extensively for the quantitation of H2O2. The diacetate form, H2DCFDA and its acetomethyl ester H2DCFDA-AM are taken up by cells where non specific cellular esterases act upon it to cleave off the lipophilic groups, resulting in a charged compound believed to be trapped inside the cell. Oxidation of H2DCF by ROS converts the molecule to 2’, 7’ dichlorodihydrofluorescein (DCF), which is highly fluorescent. The reported wavelengths for the measurement of DCF fluorescence are 498 nm for excitation and 522 nm for emission.

To assess ROS production in glomeruli and podocytes, dihydroethidium (DHE, Invitrogen) and 2',7'‐dichlorodihydrofluorescein diacetate (H2‐DCFDA, Beyotime, China) assays were conducted.

Rhein standard (purity > 98%) was purchased from Sangon Biotech (Shanghai, China). Fmoc‐amino acids were obtained from GL Biochem (Shanghai, China). All other materials used to synthesis Rh‐GFFYE and the other two compounds were obtained from Alfa (Beijing, China). Dulbecco's modified eagle medium (DMEM), penicillin/streptomycin were obtained from Life Technologies (Carlsbad, CA, USA). Calf serum was purchased from FuHeng BioLogy (Shanghai, China).H₂DCF‐DA was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 4′,6‐Diamidino‐2‐phenylindole (DAPI) was purchased from Amy Jet Scientific Inc Co., Ltd. (Wuhan, China).

Reactive oxygen species level was determined using 2, 7-dichlorodihydrofluorescein diacetate (H₂DCF-DA; Beyotime Institute of Biotechnology, Haimen, China) according to the instructions for users. In brief, around 1 × 10⁶ cells were collected and washed with PBS, and then treated with 10 μM 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, the intensity of fluorescence was normalized with the total protein content.

Ghrelin was purchased from Sigma Chemical (St. Louis, MO, USA) and dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Gibco (Grand Island, NY, USA). Medium 199 was obtained from Sigma-Aldrich (St. Louis, MO). Annexin V-FITC and propidium iodide (PI) were obtained from Becton Dickinson (Mountain View, CA, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and H₂DCFDA were obtained from Beyotime (Beyotime Institute of Biotechnology, Shanghai, China). Anti-SOD, anti-CAT, and anti-MDA antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). SOD, CAT, and MDA colorimetric kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

To assess the level of cellular stress, HConFs were inoculated into 6-well plates (4×10⁴ cells-1). HConFs were treated with different treatments for a period of time and washed twice with PBS. The 500μl reaction solution at a concentration of 5x10^-6m H₂DCF-DA (Beyotime,1;1000) or MitoSOX Red (5μM, Invitrogen, M36008) for mitochondria-derived ROS evaluation) was incubated in the dark for 20 min at 37 °C. Next, HConFs were trypsinized and resuspended in 150μl of phosphate buffer solution (PBS). Finally, the oxidation of H₂DCF-DA and MitoSOX Red were detected by flow cytometry and analyzed by Flow-Jo_V10 software.