2 7 dichlorodihydrofluorescein diacetate

Dulbecco’s modified Eagle medium and penicillin–streptomycin solution were purchased from GIBCO (Grand Island, NY, USA). Fetal bovine serum was provided by Hyclone (Logan, UT, USA). Monoclonal rabbit antibodies, including anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p65, anti-phospho-p65, NLRP3, and cleaved caspase-1 were obtained from Cell Signaling Technology (Boston, MA, USA). Receptor for AGE (RAGE) monoclonal rabbit antibody was purchased from AbCam (Cambridge, MA, USA). HRP-marked anti-β-actin antibody was supplied by Biorad (San Diego, CA, USA). 4′-Methoxyresveratrol (3,5-dyhydroxy-4′-methoxylstilbene) was purchased from Great Forest Biomedical (Hangzhou, China). BSA was obtained from ABCONE (Shanghai, China). KI and acetic acid were obtained from Sangon Biotech (Shanghai, China). Methylglyoxal, 2’,7’-dichlorodihydrofluorescein diacetate and other reagents were purchased from Sigma (St. Louis, MO, USA).

Chemicals and reagents were obtained from the following commercial sources:
2',7'-dichlorodihydrofluorescein diacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), RPMI 1600 and an antibiotic solution containing 10,000 units of penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per mL were purchased from Thermo Fisher Scientific, Gibco (Grand Island, NY, USA). BlockAce was purchased from DS Pharma Biomedical (Japan), Biotin-HABP (biotinylated linker protein hyaluronan, 0.25mg/mL) was purchased from Hokudo Co. (Japan), Anti-Human Collagen type 1 antibody was purchased from Rockland Immunochemicals (US), Anti-human MMP-1, anti-IgG/HRP, and Streptavidin-HRP were purchased from R&D Systems (Japan), Can Get Signal was purchased from Toyobo Co. (Japan), EZ West blue dye was purchased from Atto Corp. (Japan), Chlorogenic acid (CGA) was purchased from Cayman Chemical (US). Fluorescein-5-thiosemicarbazide and sodium hyaluronate, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay reagent kit was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

Intracellular ROS was determined using a non-fluorescent dye 2′7′-dichlorodihydrofluorescein diacetate (Sigma-Aldrich), which is oxidized by ROS generated by cells into a fluorescent dye 2′,7′-dichlorofluorescin. The control and high glucose-treated BeWo were incubated in the presence of 10 µm 2′7′-dichlorodihydrofluorescein diacetate for 20 min. The fluorescence was measured using a BD FACSAria (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

The ROS specific dye 2′,7′-dichlorodihydrofluorescein diacetate was obtained from Sigma-Aldrich. Following the cell harvesting and PBS washing, the cell treatment condition for this ROS dye was applied at 10 μM (37 °C, 30 min) in the dark, was reacted in proportion to cellular ROS amount, and became a fluorescence chemical reaction for the flow cytometry and FlowJo analysis [28 (link)]. The cells (Ca9-22, CAL 27, and HGF-1) were resuspended in PBS before the flow cytometry.

For ROS measurement, the skin tissues were homogenized in 40 mM Tris-HCl buffer (pH 7.4) and centrifuged [48 (link)]. Next, 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (Sigma-Aldrich, St Louis, MO, USA) was added to the supernatant and reacted at 37 °C. Fluorescence (Excitation wavelength: 485 nm, emission wavelength: 535 nm) was measured after 30 min (SpectraMax Gemini EM fluorometer, Molecular Devices, Sunnyvale, CA, USA).

Staining with 2′,7′-Dichlorodihydrofluorescein diacetate (Sigma Aldrich, St. Louis, MO, USA) followed by flow cytometric analysis were performed to quantify ROS formation. Four × 10⁴ cells were seeded on 12-well plates and subjected to the specific treatments. Following defined periods of time, 2′,7′-Dichlorodihydrofluorescein diacetate was added at a concentration of 10 µM prior to incubation for 10 min at 37 °C, avoiding exposure to light. Then cells were washed twice with PBS and enzymatically detached using Trypsin/EDTA. Afterwards, the cells were collected, centrifuged, and washed once with PBS prior to resuspension in PBS and flow cytometric analysis (FACSCantoTM II). For each measurement 10,000 events were recorded. Further analysis was done with FlowJo version 8.7.1.