2 7 dichlorodihydrofluorescein diacetate h2dcf da

MET, butylated hydroxytoluene, thiobarbituric acid, and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) were purchased from Merck (Germany). Phospholipid standards were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Ethanol, methanol, chloroform, acetonitrile, QuEChERS extraction method ingredients, lipids extraction ingredients, and high-purity solvents used during sample preparation for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses were purchased from Avantor (Gliwice, Poland) (>98% purity). All reagents were of analytical grade. Buffers and solutions were prepared in ultrapure water. Stock solutions of MET were prepared with 25 mg mL⁻¹ of ethanol.

6-OHDA was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). CellTiter-Glo^® 2.0 was purchased from Promega Corporation (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/Ham’s-F12) and low-glucose DMEM were purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). FastGene™ RNA Basic kit was obtained from Nippon Genetics Co., Ltd. (Tokyo, Japan), PrimeScript^™ RT master mix (Perfect Real Time) was obtained from Takara Bio (Shiga, Japan), and THUNDERBIRD^® Next SYBR^® qPCR mix was obtained from Toyobo (Osaka, Japan). 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Merck KGaA (Darmstadt, Germany), and MitoSOX™ was obtained from Thermo Fisher Scientific (Waltham, MA). Antibody against actin (Code: SC-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-JNK (p46 and p54) (Code: #4668), phospho-p38 (Code: #4511), phospho-ERK (Code: #4376), and IκBα (Code: #4814) and goat anti-rabbit IgG, HRP-linked antibody (Code: #7074) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). HRP-conjugated donkey anti-mouse IgG was purchased from GE Healthcare (Tokyo, Japan).

Ergothioneine was kindly provided by Tetrahedron (Paris, France). 6-OHDA was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Ergo-koji was provided by Euglena, Co., Ltd. (Tokyo, Japan). The ergothioneine content in Ergo-koji is 73.6 mg/100 g. Ergo-koji is fermented with euglena powder (Euglena Co., Ltd., Tokyo, Japan) in the fermentation stage of Rice-koji (Sakichi Co., Ltd., Nagasaki, Japan). The addition of euglena powder ensures a higher ergothioneine content of Ergo-koji than fermentation without euglena powder. MPP+ was purchased from Cayman Chemical (Ann Arbor, MI, USA). CellTiter-Glo^® 2.0 was purchased from Promega Corporation (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/Ham’s-F12) was purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Merck KGaA (Darmstadt, Germany). The FastGene™ RNA Basic kit was obtained from Nippon Genetics Co., Ltd. (Tokyo, Japan), PrimeScript^™ RT master mix (Perfect Real Time) was obtained from Takara Bio (Shiga, Japan), and THUNDERBIRD^® Next SYBR^® qPCR mix was obtained from Toyobo (Osaka, Japan).

To assay the capacity of C. mas L. fruit extract to generate intracellular level of reactive oxygen species in keratinocytes (HaCaT) and fibroblasts (HDF) cells, the method of Grauzdytė et al. [81 (link)] with some modifications, was used. In this method, the fluorogenic dye 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; (Sigma Aldrich, Sant Louis, MO, USA) was used. Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and initially cultured before the experiment for 24 h. After this time, the DMEM medium was removed and cells (HaCaT and HDF) were treated with C. mas L. extracts dissolved in DMEM at concentrations of 1, 5, 10% for another 24 h. Then, DMEM medium with extracts was changed on 10 µM H₂DCFDA in serum-free DMEM medium in each well. Then, 5 mM hydrogen peroxide (H₂O₂; in a final concentration of 500 µM) dissolved in DMEM without serum was immediately added to the tested samples and the positive sample. After 60 min of incubation, the measurement was taken at an excitation wavelength of λ = 485 nm and an emission wavelength of λ = 530 nm using a microplate reader (FilterMax F5, Thermo Fisher Scientific, Waltham, MA, USA). This analysis included three independent experiments, with each sample being tested in triplicate.

Cells were seeded in opaque 96-well plates (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 °C and 5% CO₂ for 24 h. Then, cells were treated with 2 U/mL CLytA-DAAO and 1 mM D-Ala or 600 μM H₂O₂ for 20–120 min. Along with the treatment, 10 μg/mL 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Sigma-Aldrich, St. Louis, MO, USA) were added to cells. H2DCF-DA is a probe able to emit fluorescence when is bound to free radicals inside cells. For each treatment time used, a control containing only the probe was added. After the different treatment times, medium was removed, and plates were washed with PBS. Finally, the plate reader POLARstar Omega (BMG Labtech, Ortenberg, Germany) was used to measure fluorescence using an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

Organic solvents for preparing the extract, such as dichloromethane, methanol, and DMSO, labels such as Thiazolyl Blue Tetrazolium Bromide (MTT), Crystal Violet, Hoechst 33342, and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), and N-acetyl-L-cysteine (NAC), 4-phenylbutyric acid (4-PBA), chloroquine (Chlo), necrostatin-1 (Nec-1), and necrosulfonamide (Nsa) compounds were purchased from Sigma-Aldrich (Europe). Z-VAD-FMK (zVAD) was obtained from APExBIO Technology, United States.