The HK-2 cells were lysed in lysis buffer (BestBio) and centrifuged (12,000 ×g, 4 ℃) for 20 min. Adenosine triphosphate (ATP) activity in the cell supernatant was detected using the ATP Bioluminescent Assay Kit (Promega Corporation) in accordance with the manufacturer’s instructions. To examine intracellular reactive oxygen species (ROS), 10 µΜ of dichloro-dihydro-fluorescein diacetate (Sigma-Aldrich) was used to treat the HK-2 cells at 37 ℃ for half an hour in the dark and the cells were then washed thrice with serum-starved RPMI-1640. The results were photographed with a fluorescence microscope (magnification ×200).
Dichlorodihydrofluorescein diacetate
Manufactured by Merck Group
Sourced in United States, Germany
Dichlorodihydrofluorescein diacetate is a fluorogenic compound used as a detection reagent in various laboratory applications. It is a non-fluorescent molecule that can be converted into a highly fluorescent compound upon intracellular esterase activity or oxidation, allowing for the measurement of cellular processes such as oxidative stress.
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Lab products found in correlation
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (3 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (3 mentions)
Gallic acid, by Merck Group (3 mentions)
L ascorbic acid, by Merck Group (3 mentions)
Potassium persulfate, by Scharlab (3 mentions)
Folin ciocalteu reagent, by Kemika (3 mentions)
Methanol, by Kemika (2 mentions)
Iron 3 chloride fecl3, by Kemika (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
2 7 dichlorofluorescin diacetate, by Merck Group (2 mentions)
Atp bioluminescent assay kit, by Promega (1 mentions)
Lysis buffer, by BestBio (1 mentions)
Nahco3, by Kemika (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
1100 hplc, by Agilent Technologies (1 mentions)
Six well plate, by Corning (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
16 protocols using dichlorodihydrofluorescein diacetate
1
ATP and ROS Measurement in HK-2 Cells
2
Antioxidant Assays and Zebrafish Analyses
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The fatty acids methyl esters (FAMEs) were purchased from Supelco Co. (Bellefonte, PA, USA). The standards of L-ascorbic acid (≥99%), gallic acid (>97.5%), DPPH (2,2-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-tripyridyl-S-triazine, ≥98%), ABTS (diammonium salt of 2,2′-azino-bis(3-ethylbenzthiazolin-6-yl)sulfonic acid, >99.0%), and dichloro-dihydro-fluorescein diacetate (≥97%, DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Organic solvents (dimethyl sulfoxide (DMSO, p.a.), ethanol (p.a.), methanol (p.a.)), hydrochloric acid (HCl, p.a.), iron (III) chloride (FeCl3, p.a.), Folin–Ciocalteu reagent and NaHCO3 (p.a.) were obtained from Kemika (Zagreb, Croatia), while potassium persulfate (>98%) was purchased from Scharlab (Barcelona, Spain). Hydrogen peroxide (H2O2, 30%) was obtained from Alkaloid Skopje (Skopje, North Macedonia).
Acetonitrile with 0.1% (v/v) formic acid and water with 0.1% (v/v) formic acid, both hypergrade for HPLC-MS LiChrosolv®, were purchased from Supelco Co. (Bellefonte, PA, USA).
Zebrafish D. rerio adults of wild-type WIK strain were obtained from the European Zebrafish Resource Center of the Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Used solvents were of HPLC grade and were obtained from J.T. Baker (Bridgewater, NJ, USA).
Organic solvents (dimethyl sulfoxide (DMSO, p.a.), ethanol (p.a.), methanol (p.a.)), hydrochloric acid (HCl, p.a.), iron (III) chloride (FeCl3, p.a.), Folin–Ciocalteu reagent and NaHCO3 (p.a.) were obtained from Kemika (Zagreb, Croatia), while potassium persulfate (>98%) was purchased from Scharlab (Barcelona, Spain). Hydrogen peroxide (H2O2, 30%) was obtained from Alkaloid Skopje (Skopje, North Macedonia).
Acetonitrile with 0.1% (v/v) formic acid and water with 0.1% (v/v) formic acid, both hypergrade for HPLC-MS LiChrosolv®, were purchased from Supelco Co. (Bellefonte, PA, USA).
Zebrafish D. rerio adults of wild-type WIK strain were obtained from the European Zebrafish Resource Center of the Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Used solvents were of HPLC grade and were obtained from J.T. Baker (Bridgewater, NJ, USA).
3
Quantifying Cellular Oxidative Stress
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Cells were incubated with ee-As4S4 for 0.5–72 hrs. Following incubation, cells were collected and washed with PBS, incubated in 300 μL 10 μM 2ʹ, 7ʹ-dichlorodihydrofluorescein diacetate (Sigma-Aldrich) for 30 mins at 37°C. Afterward, cells were washed by PBS and suspended in 100 μL PBS for flow cytometer analysis.
4
Mitochondrial Dysfunction and ROS Measurement
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Cells were seeded into six-well plates (Corning, Inc.) and treated with 100 μM BDMC for 24 h. Cells were then washed with phosphate-buffered saline (PBS), and incubated in PBS containing 10 μM fluorescent probe, dichlorodihydrofluorescein diacetate (Sigma-Aldrich) for 30 min at 37°C prior to determination of reactive oxygen species (ROS) levels and then analyzed by FCM. JC-1 was used to determine the inner mitochondrial membrane potential (Δψm). Cells were incubated in RPMI-1640 (Gibco-BRL) containing cationic carbocyanine dye, JC-1 (Sigma-Aldrich; 5 μg/ml), for 30 min at 37°C then analyzed by FCM. The Δψm was calculated as the red/green fluorescent ratio. The adenosine triphosphate (ATP) concentration in the mitochondrial fraction and the cytochrome c (Cyt c) levels in the mitochondrial and cytosolic fractions of the cells were measured using reverse-phase high-performance liquid chromatography (HPLC 1100; Agilent Technologies, Palo Alto, CA, USA). The translocation of Cyt c was assessed using its ratio in the mitochondrial and cytosolic fractions following the manufacturer’s instructions.
5
Intracellular ROS Measurement by DCFH-DA
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Intracellular ROS were measured by following the conversion of the nonfluorescent dichlorodihydrofluorescein diacetate (Sigma Aldrich, Milan, Italy) into a highly fluorescent compound, dichlorofluorescein, by monitoring the cellular esterase activity in the presence of peroxides as previously described [32 (link)]. The ROS generation was assessed by inducing the uptake of 1 μM nonfluorescent dichlorodihydrofluorescein diacetate and incubating for 10 min at room temperature in the dark, followed by flow cytometric analysis (CytoFLEX BeckmanCoulter, Brea, CA, USA). The results are expressed as percentage of ROS positive cells, stressed with cigarette smoke extract.
6
Evaluating Cisplatin Resistance in HepG2 Cells
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HepG2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HepG2/DDP cells were obtained from the Cell Bank, Chinese Academy of Sciences, Shanghai, People’s Republic of China. Cisplatin, UA, and dichloro-dihydro-fluorescein diacetate were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Cell Counting Kit 8 (CCK8), JC-1 dye, and BCA Protein Assay Kit were obtained from Beyotime (Nantong, People’s Republic of China). Annexin V Fluorescein Isothiocyanate Apoptosis KGA107 Detection Kit was purchased from KeyGene (Nanjing, People’s Republic of China). Dulbecco’s Modified Eagle’s Medium and fetal bovine serum were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidene difluoride (PVDF) membrane was obtained from EMD Millipore (Billerica, MA, USA). Anti-Nrf2, anti-HO-1, anti-NQO1, anti-GST, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, and horseradish-peroxidase-conjugated secondary antibody were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
7
Tangeretin Effects on Cell Viability and Oxidative Stress
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Tangeretin (TAN) was purchased from Cayman Chemicals (Cay10009911-100). Fetal bovine serum (Gibco), DMEM, PMA (phorbol 12-myristate 13-acetate), penicillin-streptomycin, Trypsin, DAPI (4′,6-diamidino-2-phenylindole), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), dichlorodihydro-fluorescein diacetate (H2 DCFDA), Rhodamine 123, Hoechst was purchased from Sigma-Aldrich, St. Louis, MO, USA. Other chemicals and reagents used for the experiment but not mentioned here were purchased from the highest grade available used for molecular studies. The complete list of the primary and secondary antibodies used in this study is provided in Supplementary Tables S1 and S2 .
8
Quantitative Antioxidant Assay Protocols
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The standards of gallic acid (>97.5%), L-ascorbic acid (≥99%), DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (diammonium salt of 2,2′-azino-bis(3-ethylbenzthiazolin-6-yl)sulfonic acid, >99.0%), TPTZ (2,4,6-tripyridyl-S-triazine, ≥98%), dichloro-dihydro-fluorescein diacetate (≥97%, DCF-DA), AAPH (2,2-azobis (2-methylpropionamidine) dihydrochloride, 97%) and 2′,7′-dichlorofluorescin diacetate were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Dimethyl sulfoxide (DMSO, p.a.), methanol (p.a.), ethanol (p.a.), iron (III) chloride (FeCl3, p.a.), hydrochloric acid (HCl, p.a.), NaHCO3 (p.a.) and Folin–Ciocalteu reagent were obtained from Kemika (Zagreb, Croatia). Hydrogen peroxide (H2O2, 30%) was purchased from Alkaloid (Skopje, Macedonia) and potassium persulfate (>98%) from Scharlau, (Regensburg, Germany).
The solvents used for SPE were of HPLC grade and were obtained from J.T. Baker (New Jersey, PA, USA).
Acetonitrile with 0.1% (v/v) formic acid and water with 0.1% (v/v) formic acid, both hypergrade for HPLC-MS LiChrosolv®, were purchased from Supelco Co. (Bellefonte, PA, USA).
Dimethyl sulfoxide (DMSO, p.a.), methanol (p.a.), ethanol (p.a.), iron (III) chloride (FeCl3, p.a.), hydrochloric acid (HCl, p.a.), NaHCO3 (p.a.) and Folin–Ciocalteu reagent were obtained from Kemika (Zagreb, Croatia). Hydrogen peroxide (H2O2, 30%) was purchased from Alkaloid (Skopje, Macedonia) and potassium persulfate (>98%) from Scharlau, (Regensburg, Germany).
The solvents used for SPE were of HPLC grade and were obtained from J.T. Baker (New Jersey, PA, USA).
Acetonitrile with 0.1% (v/v) formic acid and water with 0.1% (v/v) formic acid, both hypergrade for HPLC-MS LiChrosolv®, were purchased from Supelco Co. (Bellefonte, PA, USA).
9
Measuring Cellular ROS Levels
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Dichlorodihydrofluorescein diacetate (Sigma‒Aldrich) can be used to mark the expression level of cellular ROS. Cultures of human GCs were spiked with 10 μM Dichlorodihydrofluorescein diacetate for 30 min, followed by three washes of human GCs with PBS before capturing the fluorescence signal using a fluorescence microscope (Leica).
10
Hydrogen Peroxide Detection in Cells
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After 48 hours of treatment with ATO ± Fenton-like molecule, the supernatant was removed and 50 µL per well of 50 µg/mL 2’, 7’-dichlorodihydrofluorescein di-acetate (Sigma-Aldrich, Saint-Quentin- Fallavier, France) in PBS was added.
The H2O2 production was assessed by spectrofluorimetry using a Fusion spectrofluorometer (Packard). Fluorescence intensity was recorded immediately (T0 hours) and after 6 hours of incubation (T6 hours). Fluorescence excitation/emission maxima were for 2’, 7’-dichlorodihydrofluorescein diacetate at wavelengths of 485 and 530 nm in arbitrary units (AU).
The H2O2 production was assessed by spectrofluorimetry using a Fusion spectrofluorometer (Packard). Fluorescence intensity was recorded immediately (T0 hours) and after 6 hours of incubation (T6 hours). Fluorescence excitation/emission maxima were for 2’, 7’-dichlorodihydrofluorescein diacetate at wavelengths of 485 and 530 nm in arbitrary units (AU).
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