Dichloro-dihydro-fluorescein diacetate (H2DCFH-DA; Sigma, Darmstadt, Germany) was used at a concentration of 10 µM to determine overall ROS in the cells. Fluorescence was measured at Ex 485 nm/Em 520 nm.
H2dcfh da
Manufactured by Merck Group
Sourced in United States, Germany
H2DCFH-DA is a fluorescent dye used for the detection of reactive oxygen species (ROS) in biological systems. It is a cell-permeant indicator that is non-fluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pi dye, by Merck Group (2 mentions)
Valinomycin, by Merck Group (2 mentions)
Spectramax m5 microplate reader, by Molecular Devices (2 mentions)
Lsr fortessa facs analyzer, by BD (1 mentions)
Deferoxamine, by Merck Group (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Resazurin, by Merck Group (1 mentions)
β actin d6a8, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Tween 20, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
13 protocols using h2dcfh da
1
Measurement of Cellular Oxidative Stress
2
Cell Viability and Apoptosis Assay
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MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], H2-DCFH-DA), DMSO, Annexin V FITC, and PI dye were bought from Sigma-Aldrich (St. Louis, Missouri, United States). DMEM, fetal bovine serum (FBS), and antibiotics were bought from Gibco, USA.
3
Quantifying Mitochondrial and Oxidative Stress
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To quantify the modification of the mitochondrial membrane potential (MMP) and the reactive oxygen species (ROS) production in CCRF-CEM cells resulting from the application of BTL, either compound 8 or DMSO (negative control), or the respective positive controls for MMP or ROS evaluations, valinomycin or H2O2 (Sigma-Aldrich, Taufkirchen, Germany), the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Biomol, Hamburg, Germany) staining and the 2´,7´-dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich) staining, respectively for MMP and ROS measurements, were combined with the flow cytometry used in similar experimental conditions, as earlier reported [45 (link)]. The concentrations of samples used to treat the CCRF-CEM cells were 0.25, 0.5, 1, and 2 folds the IC50. The treated cells (1 mL; 1 × 106 cells) were incubated for 24 h under the standard cell culture condition. Cells were further stained for 30 min with 10 µL of staining solution (JC-1 for ROS evaluation or H2DCFH-DA solution for ROS evaluation) according to the manufacturer’s protocol. Using the LSRFortessa FACS analyzer (Becton–Dickinson, Heidelberg, Germany), the amount of 1 × 104 cells was further measured as described earlier [31 (link), 45 (link)–47 (link)].
4
ROS Measurement in MCF7 Cells
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The MCF7 cells (3 ml, 1 × 104 cells/ml) treated for 24 h (humidified 5% CO2 atmosphere at 37 °C) with different concentrations (¼ × IC50, ½ × IC50 and IC50) of compound 1 , and doxorubicin (drug control) or DMSO (solvent control)) were analyzed for ROS production with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma–Aldrich) using OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) as recommended by the manufacturer, Cell Biolabs Inc. (San Diego, USA) (Kuete et al., 2016). The fluorescence was measured using SpectraMax® M5 Microplate Reader (Molecular Devices, Biberach, Germany) at 480/530 nm. All experiments were performed in triplicates.
5
Apoptosis and Oxidative Stress Evaluation
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Solvents such as n-hexane, chloroform, ethyl acetate, butanol, acetonitrile, and methanol were purchased from Chem-Lab (Zedelgem, Belgium). Penicillin and streptomycin were obtained from Gibco (Co Dublin, Ireland). Annexin V-FITC/PI detection apoptosis kit was obtained from Life Technologies (Carlsbad, CA, USA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) kits from Biomol (Hamburg, Germany). H2DCFH-DA, H2O2, ferrostatin-1, deferoxamine, valinomycin, PI, resazurin, triton X-100, paraformaldehyde, tween-20, dimethylsulphoxide (DMSO), DAPI and doxorubicin (98.0% purity) were purchased from Sigma-Aldrich (Taufkirchen, Germany). M-PER® mammalian protein extraction reagent and protease inhibitor were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against Beclin-1 (D40C5), LC3B, β-actin (D6A8) and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Technology (Danvers, MA, USA) and Luminata™ Classico Western HP substrate was obtained from Merck Millipore (Darmstadt, Germany).
6
Lanthanum Oxide Nanoparticle Cytotoxicity
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Lanthanum oxide (La2O3) nanopowder (La2O3, 99.99%, 10–100 nm, Stock#: US3265) was purchased from US Research Inc. Houston TX USA. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], H2-DCFH-DA), DMSO, Annexin V-FITC, and PI dye were bought from Sigma-Aldrich (St. Louis, Missouri, United States). DMEM, fetal bovine serum (FBS), and antibiotics were bought from Gibco, USA.
7
ROS Assay for CCRF-CEM Cells
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Various concentrations of HRB and triterpenoid 1 were used to treat CCRF-CEM cells (1 × 106 cells); DMSO (solvent control); or hydrogen peroxide (H2O2; positive control). After 24 h incubation in humidified 5% CO2 atmosphere at 37°C, the production of ROS was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich) staining, as described earlier [30 –32 (link)].
8
ROS Induction in MCF-7 Cells
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The MCF-7 cells were treated with compounds 9 and 13 and doxorubicin, and ROS production was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich) by OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) as recommended by the manufacturer, Cell Biolabs Inc. (San Diego, USA) [23 (link)]. Cells were tested in 6-well plates (3 mL, 1 × 105 cells/mL) and the incubation time was 24 h in humidified 5% CO2 atmosphere at 37 °C. The tested concentrations were ¼ × IC50, ½ × IC50 and IC50. Untreated cells (control) were used for comparison with treated cells. The fluorescence was measured using SpectraMax® M5 Microplate Reader (Molecular Devices, Biberach, Germany) at 480/530 nm. All experiments were performed at least in triplicates.
9
Quantifying Cellular Oxidative Stress
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2′,7′-Dichlorofluorescin diacetate (H2DCFH-DA, Sigma-Aldrich) is a cell-permeable non-fluorescent probe used to detect cellular ROS levels. In the presence of ROS, the compound is de-esterified intracellularly and converts into the highly fluorescent 2′,7′-dichlorofluorescein upon oxidation. Thus, it can be measured by flow cytometry [67 (link)]. CCRF-CEM cells were re-suspended in PBS and incubated for 30 min withH2DCFH-DA at a concentration of 2 μM. After washing with PBS, the cells were treated with DMSO as negative control, H2O2 and doxorubicin as positive controls or varying concentrations of Aloe-emodin (0.5-, 1-, 2- and 4-fold IC50) for 1 h. The results were assessed by a BD Accuri™ C6 flow cytometer (Becton Dickinson) using FL-1 the channel (488 nm excitation). For each sample, 104 cells were counted. The protocol has been recently reported by us [68 (link)].
10
Synthesis and Characterization of La2O3 Nanoparticles
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Lanthanum oxide (La2O3) nanoparticles (La2O3NPs, 99.99%, 10–100 nm, Stock#: US3265) was purchased from US Research Inc. Houston TX USA. MTT, H2-DCFH-DA), DMSO, and Annexin V FITC were bought from Sigma-Aldrich (St. Louis, Missouri, United States). DMEM and fetal bovine serum were purchased from Gibco, USA.
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