All synthetic oligonucleotides were purchased from Eurofins. PCR primers were desalted, DNAzymes were purified by Eurofins proprietary High Purity Salt Free reverse phase cartridge purification system and short RNA substrates were purified by HPLC. RNase-free ultrapure water was produced using a Milli-Q Advantage A10 Water Purification System equipped with a BioPak® Polisher from Merck Millipore. Phusion High-Fidelity PCR kit and TranscriptAid T7 High Yield Transcription kit were from Life Technologies. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), sodium chloride, manganese chloride tetrahydrate, formamide, bromophenol blue, 40% 19:1 acrylamide/bis-acrylamide, N,N,N′,N′-tetramethylethylenediamine, ammonium persulfate, urea, agarose, ethidium bromide and phenol:chloroform:isoamyl alcohol 25:24:1 pH 8.0 was purchased from Sigma-Aldrich. Ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) were from Scharlau. Boric acid was from VWR. Klorrent bleach was from Nilfisk. The nucleic acid dyes SYTO 61, PO-PRO 1, DRAQ5, Hoechst 33258, DAPI, Propidium iodide and PicoGreen were all from Life Technologies. GelRed was from Biotium. ethidium bromide was from Sigma.
Ethidium bromide
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, India, Poland, Japan, Italy, China, Switzerland, Sao Tome and Principe, Spain, Canada, France, Brazil, Angola, Macao, Slovakia, Saudi Arabia
Ethidium bromide is a fluorescent dye commonly used in molecular biology laboratories. It is used to visualize and detect the presence of nucleic acids, such as DNA and RNA, in agarose gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Acridine orange, by Merck Group (128 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (61 mentions)
Agarose, by Merck Group (51 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (51 mentions)
Triton x 100, by Merck Group (41 mentions)
Trizol reagent, by Thermo Fisher Scientific (33 mentions)
Propidium iodide, by Merck Group (32 mentions)
Agarose gel, by Merck Group (32 mentions)
Sodium hydroxide (naoh), by Merck Group (25 mentions)
Bovine serum albumin (bsa), by Merck Group (24 mentions)
Sodium chloride (nacl), by Merck Group (24 mentions)
Trizol, by Thermo Fisher Scientific (23 mentions)
Fetal bovine serum (fbs), by Merck Group (21 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (20 mentions)
Streptomycin, by Thermo Fisher Scientific (20 mentions)
Penicillin, by Thermo Fisher Scientific (19 mentions)
Uridine, by Merck Group (16 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (16 mentions)
Trizma base, by Merck Group (14 mentions)
Hoechst 33342, by Merck Group (14 mentions)
928 protocols using ethidium bromide
1
Oligonucleotide Synthesis and Purification Protocols
2
Imidazolium Amphiphile Synthesis and Characterization
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Imidazolium amphiphile IA-12 was synthesized in accordance with the standard experimental procedure [45 (link),49 (link)]. Commercially available double-stranded oligonucleotide (oNu) with 10 nucleotide units in each strand (GCGTTAACGC, molecular weight is 3028 g/mol, Joint Stock Company Syntol, Moscow, Russia) was used. Plasmid DNA (pDNA) pVax1-DsRed (3665 bp) was kindly gifted by Dilara Gatina (Omics Technology Laboratory, Kazan Federal University, Kazan, Russia). Agarose for molecular biology, ethidium bromide and hexadecyltrimethylammonium bromide (≥99.0%) were purchased from Sigma-Aldrich. As probes for fluorometric measurements, ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA, 95%) and pyrene (Sigma-Aldrich, St. Louis, MO, USA, 99%) were used. Lipoid PC 16:0/16:0 (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) was a gift from Lipoid GmbH (Ludwigshafen, Germany). Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA, 99%) was applied for the fluorescence microscopy assay. Polyacrylic acid with an average molecular weight of 1800 g/mol (Sigma-Aldrich, St. Louis, MO, USA, 99%) was used. Polymer concentrations were given as a molar concentration on a monomer basis. Purified water (18.2 MΩ cm resistivity at 25 °C) from Direct-Q 5 UV equipment (Millipore S.A.S. 67120 Molsheim-France) was used for all solution preparation.
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Cytotoxic Effects of Methanol and Ethidium Bromide on AGS Cells
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Methanol and ethidium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Sigma Chemical Co. (USA). Stock cultures of AGS-CRL-1739 human stomach cancer cells were purchased from the American Type Culture Collection (USA). Ham's F-12 K (Kaighn's) Medium and fetal bovine serum (FBS) used in a cell culture were products of Gibco (USA). Glutamine, penicillin, and streptomycin were obtained from Quality Biologicals Inc. (USA); [3H]thymidine (6.7 Ci mmol−1) was purchased from NEN (USA), and Scintillation Cocktail “Ultima Gold XR” from Packard (USA). Sodium dodecyl sulfate was received from Bio-Rad Laboratories (USA). Acridine orange and ethidium bromide were provided by Sigma Chemical Co. (USA). FITC Annexin V Apoptosis Detection Kit II was product of BD Pharmingen.
4
Ethidium Bromide-Mediated Mitochondrial DNA Depletion
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Cells were treated with ethidium bromide (150 ng/ml; Sigma-Aldrich) for 6 d. During ethidium bromide treatment, cells were maintained in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 µg/ml uridine. To measure mtDNA depletion efficiency, cellular DNA was isolated using QIAamp DNA mini kit, and mitochondrial ND4 gene was quantified using real-time PCR and normalized to genomic GAPDH gene. The primer pair used is as following: ND4 forward, 5′-AACGGATCCACAGCCGTA-3′; ND4 reverse, 5′-AGTCCTCGGGCCATGATT-3′. mtDNA-depleted cells were replated, cultivated overnight, and total RNA was extracted for ISGs expression measurement by real-time RT-PCR.
5
Plasma Membrane Permeability Dynamics
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Changes in plasma membrane permeability after exposure to BzATP (500 μM) (Sigma‐Aldrich) were studied by ethidium bromide uptake. IVD cells were kept at 37°C in a thermostat‐controlled and magnetically stirred cuvette of a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) in the presence of 20 μM ethidium bromide (Sigma‐Aldrich). Fluorescence changes were acquired at 360 and 580 nm excitation and emission wavelengths, respectively. Full permeabilization was obtained with 100 μM digitonin.
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Evaluating Cell Membrane Permeability to BzATP
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Changes in plasma membrane permeability on exposure to BzATP (200 μM) (Sigma-Aldrich) were studied by ethidium bromide uptake. One hundred thousand cells were kept at 37° C in a thermostat-controlled and magnetically stirred cuvette of a Cary Eclipse Fluorescence Spectophotometer (Agilent Technologies) in the presence of 20 μM ethidium bromide (Sigma-Aldrich). Fluorescence changes were acquired at 360 nm and 580 nm excitation and emission wavelengths, respectively.
For A172, M059J, T98G, U87MG, 8 × 104 cells were seeded in 96-well plates for 24 h, washed with PBS, incubated for 10 min in PBS supplemented with ethidium bromide (5 μg/mL) and 200 μM BzATP. A positive control of ethidium bromide incorporation was performed treating cultures with permeable buffer solution during ethidium bromide exposure (3.5 mM trisodium citrate, 0.5 mM Tris buffer and 0.1 % nonidet). ethidium bromide fluorescence was observed using a inverted fluorescence microscope (Nikon Eclipse TE300; Melville, NY, USA). The exposure time used to acquire images from treated samples was the same used for acquisition of images from permeabilized cells (maximal ethidium bromide uptake).
For A172, M059J, T98G, U87MG, 8 × 104 cells were seeded in 96-well plates for 24 h, washed with PBS, incubated for 10 min in PBS supplemented with ethidium bromide (5 μg/mL) and 200 μM BzATP. A positive control of ethidium bromide incorporation was performed treating cultures with permeable buffer solution during ethidium bromide exposure (3.5 mM trisodium citrate, 0.5 mM Tris buffer and 0.1 % nonidet). ethidium bromide fluorescence was observed using a inverted fluorescence microscope (Nikon Eclipse TE300; Melville, NY, USA). The exposure time used to acquire images from treated samples was the same used for acquisition of images from permeabilized cells (maximal ethidium bromide uptake).
7
Immortalized Human Colon Cell Line NCM460
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The human immortalized colon cell line NCM460 was acquired from Procell Company. (Wuhan, China). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher, United States) with 10% fetal bovine serum (Gibco, United States) and 1% pen-strep (100 U/ml penicillin and 100 mg/ml streptomycin) (Gibco, United States). The mtDNA copy number knockdown was induced by maintaining in 50 ng/ml ethidium bromide (EB) with 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, United States) and 50 μg/ml uridine (Sigma-Aldrich, St. Louis, MO, United States) ethidium bromide (EB) maintained for over 4 months.
8
Ethidium Bromide Accumulation and Efflux Assays
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The ethidium bromide accumulation and efflux assays were measured by florescence intensity (48 (link), 89 (link)) with minor modifications. Briefly, mid-log-phase cultures were washed with PBS containing 0.05% Tween 80 (PBST) and then stained with 2 µg/ml ethidium bromide (Sigma). ethidium bromide (1 µg/ml) was used for accumulation assays with efflux inhibitors, including chloropromazine (10 µg/ml; Sigma), verapamil (100 µg/ml; Sigma), reserpine (6 µg/ml; Sigma), or carbonyl cyanide m-chlorophenyl hydrazone (1 µg/ml; Sigma). For the ethidium bromide efflux assay, bacteria were washed with PBST and then incubated with 2 µg/ml ethidium and 100 µg/ml verapamil for 60 min. After the bacteria were washed twice with PBST, efflux activity was measured as the decay ratio of fluorescence intensity. For Nile red uptake staining, mid-log-phase cultures were washed with PBS and then stained with 20 µM Nile red (Sigma) (90 (link)). In all assays, the cells were incubated in 96-well plates, and analysis was performed at the indicated time points by excitation at 544 nm and emission at 590 nm on a FLUOstar OPTIMA microplate reader (BMG Labtech). All data were normalized to the time zero reading of each well. All experiments were repeated at least three times and similar results were obtained. Representative results are shown in Fig. 7A and B .
9
Apoptosis Induction and Cell Proliferation Assays
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Methanol and ethidium bromide,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Sigma Chemical Co. (USA). Stock cultures of AGS- CRL-1739 human stomach cancer cells were purchased from the American Type Culture Collection (USA). Ham’s F-12 K (Kaighn’s) Medium and fetal bovine serum (FBS) used in a cell culture were products of Gibco (USA). Glutamine, penicillin and streptomycin were obtained from Quality Biologicals Inc. (USA). [3H]thymidine (6.7 Ci mmol−1) was purchased from NEN (USA), and Scintillation Coctail “Ultima Gold XR” from Packard (USA). Sodium dodecyl sulfate was received from Bio-Rad Laboratories (USA). Acridine orange and ethidium bromide were provided by Sigma Chemical Co (USA). FITC Annexin V Apoptosis Detection Kit II was a product of BD Pharmigen. Topoisomerase II Drug Screening Kit was a product of TopoGEN (Florida, USA).
10
2D Neutral-Neutral Agarose Gel Electrophoresis
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Neutral-neutral 2D agarose gel electrophoresis was performed as described (Zhang et al., 2017 (link)). Briefly, 10 µg genomic DNA was extracted (Qiagen) and digested with restriction enzymes RsaI (NEB) and HinfI (NEB), to which Exonuclease I (NEB) was added 2 h before restriction enzyme treatment to remove 3′ overhang when appropriate. Digested genomic DNA was subjected to first dimensional gel using 0.4% agarose gel in 1× TBE buffer and electrophoresed at 1 V/cm for 12 h at room temperature. Sample lanes were excised and immersed in 0.3 µg/ml ethidium bromide (Sigma-Aldrich) for 30 min and casted into 1% agarose gel with 0.3 µg/ml ethidium bromide in 1× TBE. The secondary dimensional gel was electrophoresed at 3 V/cm for 6 h at 4°C. Afterward, the gel was pumped to dry using gel dryer (Bio-Rad) and subjected to native and denatured in gel hybridization with 32P-labeled C-probe. The gel was washed and exposed to PhosphorImager screen, which was then scanned by Storm and analyzed with Fiji.
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