The following antibodies were used in the immunoblotting, co-immunoprecipitation, and immunofluorescence assays: HA monoclonal Ab (mAb), GAPDH polyclonal Ab (pAb), TOM40 mAb, and TIM23 mAb (Santa Cruz, CA, USA); OPTN pAb (Abcam, MA, USA); β-actin mAb and Flag mAb (Sigma-Aldrich, MO, USA); and Caspase-3 mAb and PARP Ab (Cell Signaling Technology, MA, USA). Propidium Iodide (PI), Carbonyl cyanide m-chlorophenyl hydrazine (CCCP), Cycloheximide (CHX), and 2′,7′-Dichlorofluorescin diacetate (H2DCFDA) were obtained from Sigma-Aldrich.
2 7 dichlorofluorescin diacetate h2dcfda
Manufactured by Merck Group
Sourced in United States, Germany
2',7'-Dichlorofluorescin diacetate (H2DCFDA) is a fluorogenic compound used as a cell-permeant indicator for reactive oxygen species (ROS) in biological systems. It is non-fluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell, resulting in a fluorescent compound.
Automatically generated - may contain errors
Lab products found in correlation
Gallic acid, by Merck Group (3 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
N acetylcysteine nac, by Merck Group (2 mentions)
Quercetin, by Merck Group (2 mentions)
N acetylcysteine, by Merck Group (2 mentions)
Dulbecco s phosphate buffered saline dpbs, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by PAN Biotech (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Sodium alginate, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Celltiter glo 3d reagent, by Promega (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
Monochlorobimane mcb, by Merck Group (1 mentions)
Cytotoxicity detection kit, by Roche (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
20 protocols using 2 7 dichlorofluorescin diacetate h2dcfda
1
Antibody Characterization and Reagent Acquisition
2
Lipid-based Nanoparticle Characterization
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1,2-Dipalmitoylphosphatidylcholine (DPPC) was gifted from Lipoid AG (Steinhausen, Switzerland). Cholesterol (CH), PAMAM dendrimer; ethylenediamine core, generation 5.0 solution (5 wt% in methanol), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2,7-dichlorofluorescin diacetate (H2DCFDA) were obtained from Sigma Aldrich Chemie GmbH (Taufkirchen, Germany). Pierce protein BCA assay kit and Gene JET Plasmid Miniprep kit were purchased from Thermo Fischer Scientific (Dreieich, Germany). Promokine Calcein-AM and PromoKine LDH cytotoxicity kit II was purchased from Promo Cell GmbH (Darmstadt, Germany). CellTiter-Glo 3D Reagent and Cell culture lysate reagent (CCLR) was purchased from Promega Corporation (Mannheim, Germany). Dulbecco's modified Eagle's minimum essential medium (DMEM), Fetal bovine serum (FBS) were purchased from Capricon (Ebsdorfergrund, Germany). Purified water from PURELAB flex II dispenser (ELGA Lab Waters, High Wycombe, UK) was used for different experiments. All other reagents used in experiments were of analytical grade.
3
Measuring ROS and Reduced GSH in Chives
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According to the previous method [43 (link)], the ROS in chives were determined using a Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany). Briefly, the leaf apex of Chinese chive was incubated with 25 μM 2′, 7′-dichlorofluorescin diacetate (H2DCFDA; Sigma-Aldrich, Saint Louis, America, http://www.sigmaaldrich.com (accessed on 25 April 2021)) for 20 min in the dark. After washing with HEPES/NaOH buffer (pH 7.5) three times, the sample was detected immediately by confocal microscope. Detection was performed by λ (excitation) = 488 nm and λ (emission) = 500–530 nm. The relative fluorescence was expressed as values relative to the control group at 0 day.
The reduced GSH content in chives was estimated following the previous method [44 (link)]. The leaf apex of Chinese chive was incubated with 50 μM monochlorobimane (MCB; Sigma-Aldrich, Saint Louis, America,http://www.sigmaaldrich.com (accessed on 25 April 2021)) for 20 min in the dark and was washed with HEPES buffer (pH 7.5) three times. Subsequently, the sample was detected immediately by a Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany; emission at 461 nm, excitation at 380 nm, respectively). The relative fluorescence was presented as values relative to Con at 0 day.
The reduced GSH content in chives was estimated following the previous method [44 (link)]. The leaf apex of Chinese chive was incubated with 50 μM monochlorobimane (MCB; Sigma-Aldrich, Saint Louis, America,
4
In Vitro Cell Cytotoxicity Assays
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DMEM/F12 phenol red-free, trypsin, charcoal/dextran-treated fetal bovine serum (FBS), penicillin, streptomycin, glycerol, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA), DL-dithiothreitol (DTT), TBC (269999), Hoechst 33342, calcein AM, 2’,7’ –dichlorofluorescin diacetate (H2DCFDA), and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The substrate for caspase-1 and caspase-3 were purchased from Merck (Darmstadt, Germany). The cytotoxicity detection kit was purchased from Roche Applied Science (Mannheim, Germany). ELISA kits for Sod1 (M2398), IL-1β (M0037), IL-1βR1 (M0017), and Cat (M2605) were purchased from Elabscience Biotechnology (Wuhan, China). ELISA kit for Ki67 (EM1473) was purchased from Fine Biotech (Wuhan, China). Stock solutions of these compounds were prepared in DMSO and added to DMEM/F12 medium. The final concentration of DMSO in the culture medium was always 0.1%.
5
Evaluating SiO2 Nanoparticle Cytotoxicity
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SiO2 NPs were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM), and penicillin/streptomycin were obtained from Life Technologies (Carlsbad, CA, USA). N-acetylcysteine (NAC) and 2′,7′-dichlorofluorescin diacetate (H2DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A JC-1 mitochondrial membrane potential assay kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China).
6
Mitochondrial Staining and ROS Detection in HCT-116 Cells
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For mitochondrial staining, HCT-116p53+/+ and HCT-116p53−/− cells were incubated with 0.3 μM MitoTracker Red CMXRos (Invitrogen, Eugene, Oregon, USA) for 30 min at 37 °C in 5% CO2. Afterwards, cells were stained with 3 μM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) for 15 min at 37 °C in 5% CO2. For ROS measurement, HCT-116p53+/+ and HCT-116p53−/− cells were incubated with 0.1 μM 2′,7′-dichlorofluorescin diacetate (H2DCFDA, Sigma-Aldrich) for 30 min at 37 °C in 5% CO2. Afterwards, cells were stained with 3 μM DAPI for 15 min at 37 °C in 5% CO2. All assays were performed using a BD FACSAria II flow cytometer (San Jose, CA, USA), and the BD FACSDiva Software version 8.0.2. Data were analyzed with the BD FlowJo version 10 software.
7
Evaluating Cellular Stress in Insect Cells
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Fetal bovine serum (FBS) was purchased from LGC Biotecnologia (Cotia, SP, Brazil). The Schneider’s Insect Medium (SIM), Dulbecco’s Modified Eagle Medium (DMEM), and Grace’s Insect Medium (GIM), amphotericin B solution (AmB, 250 µg/mL), penicillin-streptomycin solution (5000 units penicillin and 5 mg streptomycin/mL), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), thiazolyl blue tetrazolium Bromide (MTT), 2,2′ azobis (2-methylpropionamidine) dihydrochloride (AAPH), 2′,7′-dichlorofluorescin diacetate (H2DCFDA), lipopolysaccharide (LPS), and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was purchased from Synth (Diadema, SP, Brazil). A Panoptic staining kit was obtained from Laborclin (Pinhais, PR, Brazil). All other reagents were analytical grade.
8
Cytotoxicity and Oxidative Stress in PC12 Cells
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PC12 cells (rat adrenal pheochromocytoma cells) were obtained from American Type
Culture Collection (ATCC, USA). Roswell Park Memorial Institute 1640 (RPMI-1640)
medium and foetal bovine serum (FBS) were also purchased from ATCC and
AlamarBlue cell viability assay reagent (DAL1100) was obtained from Thermo
Fisher Scientific, USA. (H2DCFDA). 2′,7′-Dichlorofluorescin diacetate
(H2DCFDA), a chemically reduced form of fluorescein used as an
indicator for ROS production, was purchased from Sigma-Aldrich, USA. The Annexin
V-FITC apoptosis staining/detection kit with propidium iodide staining solution,
was supplied by BD Biosciences, USA.
Antibodies against the mitochondrial proteins, Bax, Bcl-2, Cytochrome C,
Caspase-3 and β-actin, were obtained from Santa Cruz Biotechnology, Santa Cruz,
USA. ABTS (2,2’-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]), metal salts
and small molecules were purchased from Sigma-Aldrich, USA. All other chemicals
used in this study were all analytical grade.
Culture Collection (ATCC, USA). Roswell Park Memorial Institute 1640 (RPMI-1640)
medium and foetal bovine serum (FBS) were also purchased from ATCC and
AlamarBlue cell viability assay reagent (DAL1100) was obtained from Thermo
Fisher Scientific, USA. (H2DCFDA). 2′,7′-Dichlorofluorescin diacetate
(H2DCFDA), a chemically reduced form of fluorescein used as an
indicator for ROS production, was purchased from Sigma-Aldrich, USA. The Annexin
V-FITC apoptosis staining/detection kit with propidium iodide staining solution,
was supplied by BD Biosciences, USA.
Antibodies against the mitochondrial proteins, Bax, Bcl-2, Cytochrome C,
Caspase-3 and β-actin, were obtained from Santa Cruz Biotechnology, Santa Cruz,
USA. ABTS (2,2’-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]), metal salts
and small molecules were purchased from Sigma-Aldrich, USA. All other chemicals
used in this study were all analytical grade.
9
CeO2 NPs Cytotoxicity Evaluation
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CeO2 NPs were purchased from Shanghai Xiangtian Nanomaterials Co., Ltd. (Shanghai, China); fetal bovine serum (FBS), DME/F−12 medium, and penicillin/streptomycin were obtained from Life Technologies (Carlsbad, CA, USA); N-acetylcysteine (NAC), and 2′,7′-Dichlorofluorescin diacetate (H2DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA); and a mitochondrial membrane potential assay kit with JC-1 was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China).
10
Arecoline Hydrobromide: Detailed Protocol
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Arecoline hydrobromide (methyl 1-methyl-1, 2, 5, 6-tetrahydronicotinate hydrobromide) was obtained from Sigma-Aldrich (St. Louis, MO, USA); its purity was greater than 99.0 %. Rotenone, antimycin A, 2′,7′-dichlorofluorescin diacetate (H2DCF-DA), and BCECF AM ester were also from Sigma-Aldrich (St. Louis, MO, USA). Reduced L-glutathione was from Boehringer Mannheim GmbH (Mannheim, West Germany). Protein assay reagents were from Bio-Rad Laboratories (Hercules, CA, USA). Fluo-4 AM ester were from Molecular Probes (Eugene, OR, USA). TRIzol reagent was from Invitrogen Life Technologies (Carlsbad, CA, USA). All other chemicals, including calcium carbonate, 2-deoxy-d-glucose, and N-acetyl-L-cysteine, were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).
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