2 7 dichlorofluorescein diacetate dcfh2 da

In vitro detection of intracellular ROS on fibroblasts BALB/3T3 using DCFH₂-DA
The assay to evaluate the in vitro antioxidant activity was carried out using the murine BALB/3T3 fibroblasts that were plated in a white 96 MW and then treated with the complexes at a concentration of 25 µM for 24 h. Menadione (25 µM) was then added for 15 min to induce ROS production. After treatment, the cells were washed with PBS and treated with 25 μM 2′-7′-dichlorofluorescein diacetate (DCFH₂-DA, Sigma-Aldrich, St. Louis, MO, USA), and then they were incubated for 40 min at 37 °C and 5% CO₂. The probe DCFH₂-DA penetrates cells where it is esterified into a non-fluorescent form (DCFH₂) by endogenous esterases. In the presence of intracellular ROS, non-fluorescent H₂DCF is oxidized to the fluorescent 2′,7′-dichlorofluorescein (DCF). Then, the cells were washed with PBS and the fluorescence was detected using a multiplate reader (λ_ex = 485 nm, λ_em = 535 nm).
Based on the obtained fluorescence values, the ROS production inhibition % (I_ROS) vs. menadione was calculated using the following formula: $$I_{ROS} \mathrm{v}\mathrm{s}. Menadione (\%)=\frac{F_{Men}-F_{Sample}}{F_{Men}}\ast 100$$
where F_Men represents the mean fluorescence obtained after treatment with menadione alone and F_sample represents the mean fluorescence obtained after the co-treatment with the complexes and menadione.

Lyophilized silk sericin powder was kindly provided by Ruenmai-baimon, LTD., Surin Province, Thailand. Porcine pancreas trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), Alcalase^® (EC 3.4.21.62), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), ferric chloride hexahydrate (FeCl₃⋅6H₂O), 37% hydrochloric acid (HCl) solution, 1 M sodium hydroxide (NaOH) solution, fluorescein, 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH), sodium dodecyl sulfate (SDS), Coomassie brilliant blue R-250, isopropanol, ethanol, acetic acid solution, 2′,7′-dichlorofluorescein diacetate (DCFH₂-DA) and N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A bicinchoninic acid (BCA) protein assay kit used for determination of total protein content was procured from Thermo Scientific (Rockford, IL, USA). MilliporeSigma (Burlington, MA, USA) was the source of 3% w/w hydrogen peroxide (H₂O₂) solution.

Antrodin C was isolated from the mycelia of A. cinnamomea as described previously [26 (link)]. The purity of ADC was above 99% according to HPLC and ¹H-NMR analyses. M199 medium, fetal bovine serum (FBS), sodium pyruvate, penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA). Endothelial cell growth supplement (ECGS), heparin sodium salt, d-Glucose, 2’, 7’-dichlorofluorescein diacetate (DCFH₂-DA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), zinc protoporphyrin (ZnPP) and resveratrol (RES) were purchased from Sigma-Aldrich (St. Louis, CA). Antibodies against phos-Rb, cyclin D1, cyclin E, CDK2, CDK4, CDK6, Acetyl-p53, p16^INK4A, p21^CIP1, phos-p53, Keap-1, Pro-caspase-3, Clev-caspase-3, Pro-caspase-9, Clev-caspase-9, Pro-PARP, Clev-PARP, Cytochrome C and Bax were obtained from Cell Signaling Technology, Danvers, MA. Antibodies against HO-1, NQO-1 and Nrf2 were purchased from Abcam, Cambridge, UK. Antibodies against SMP30, p53 and β-actin were obtained from Santa-Cruz Biotechnology, Dallas, TX. All other chemicals were reagent grade or HPLC grade and supplied by either Merck (Darmstadt, Germany) or Sigma-Aldrich.

The seaweed was collected in June 2019 from the coastal area of Jeju Island, South Korea. Penicillin-streptomycin, Dulbecco’s modified Eagle medium, trypsin-EDTA, and fetal bovine serum were purchased from Gibco-BRL. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,3-bis (diphenylphosphino) propane (DPPP), acridine orange (AO), dimethyl sulfoxide, 2,7-dichlorofluorescein diacetate (DCFH2-DA), and EtOH were purchased from Sigma-Aldrich Co (St. Louis, MO, USA).

In order to detect reactive oxygen species (ROS) levels and cell integrity of the indicator bacteria after treated with purified antimicrobial agents, when P. aeruginosa PAO1 and MRSA grow to the OD₆₀₀ of 0.3, fengycin and surfactin were added to P. aeruginosa PAO1 and MRSA at the final concentration of 200 μg/ml, respectively, and incubated at 28°C for 4 h. After incubation, 2′,7′-dichlorofluorescein diacetate (DCFH₂-DA; Sigma-Aldrich) dye was added and incubated for 30 min under dark conditions, then the cells were observed under a fluorescent microscope with a filter (488 nm/525 nm). Similarly, to detect the cell integrity, P. aeruginosa PAO1 and MRSA were treated with the same concentration of fengycin or surfactin, then stained with propidium iodide (PI) dye for 30 min under dark conditions and observed under fluorescent microscope with a 535 nm/615 nm filter. The same treatment was performed on the control group, with methanol instead of the fengycin or surfactin.

Dulbecco’s modified Eagle medium (DMEM), Ham’s nutrient mixtures medium (F-12 medium), trypsin-EDTA, penicillin-streptomycin (P/S), and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). PIP ELISA kit was purchased from TaKaRa Bio Inc (Shiga, Japan). The 2,7-dichlorofluorescein diacetate (DCFH2-DA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,3-Bis (diphenylphosphino) propane (DPPP), diaminofluorophore 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM-DA), and the ELISA kits used for the analysis of human MMPs were purchased from Sigma (St. Louis, MO, USA). All other chemicals used in this study were of analytical grade.