TriZol, oligo dT18 primer, reverse transcriptase buffer, dNTPs, murine Maloney leukemia virus (MMLV) reverse transcriptase, RNase inhibitor, and agarose were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Ethidium bromide (EtBr) was purchased from Sigma (St. Louis, MO, USA), and dextran sulfate sodium (DSS; molecular weight: 36,000-50,000) from MP Biomedical (Solon, OH, USA). All chemicals used were of analytical grade.
Ethidium bromide etbr
Manufactured by Merck Group
Sourced in United States, Germany
Ethidium bromide (EtBr) is a laboratory dye used for the detection of nucleic acids, such as DNA and RNA, in gel electrophoresis. It intercalates between the base pairs of nucleic acid molecules, emitting a bright orange fluorescence when exposed to ultraviolet light. EtBr is a commonly used reagent in molecular biology and biochemistry laboratories for visualizing and analyzing nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Acridine orange, by Merck Group (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Chloroform, by Merck Group (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Ethanol, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (2 mentions)
Phenol chloroform isoamyl alcohol, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Sodium azide, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Ethyl acetate, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Reverse transcriptase buffer, by Thermo Fisher Scientific (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Oligo dt 18 primer, by Thermo Fisher Scientific (1 mentions)
34 protocols using ethidium bromide etbr
1
RNA Extraction and Reverse Transcription
2
Alkaline Comet Assay for DNA Damage
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To compare the levels of DNA damage in PRMT5 depleted cells and TDP1−/− MEFs cells transfected with FLAG-TDP1WT, FLAG-TDP1KK and vector control, were subjected to alkaline comet assays according to the manufacturer's instructions (Trevigen, Gaithesburg, MD) as described previously (10 (link),13 (link),36 (link)). Briefly, after treatment with 5 μM CPT, cells were collected and mixed with low melting agarose. Slides were immersed in lysis solution at 4°C for 1 h. After a rinse with deionized water, slides were immersed in a 4°C alkaline solution (50 mM NaOH, 1 mM EDTA, and 1% dimethyl sulfoxide) for 1 h. Electrophoresis was carried out at a constant voltage of 25 V for 30 min at 4°C. After electrophoresis, slides were neutralized in 0.4 M Tris–HCl (pH 7.5), dehydrated in ice-cold 70% ethanol for 5 min, and air-dried. DNA was stained with ethidium bromide (EtBr) purchased from Sigma (USA). The relative length and intensity of EtBr-stained DNA, tails to heads, is proportional to the amount of DNA damage present in the individual nucleus. Comet length was measured using the TriTek Comet Score software (TriTek Corp, Sumerduck, VA) and was scored for at least 50 cells. Distributions of comet lengths were compared using the Student t-test.
3
Antioxidant Evaluation of Zinc Acetate
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Zinc acetate dehydrade [Zn (CH3COO)2]. 2 H2O, Sodium hydroxide (NaOH), 2,2-Diphenyl-1-picrylhydrazyl assay (DPPH), 80% Methanol, Butylated hydroxyl anisole, 2, 2΄ Azinobis (3-ethyl benzo-thizoline-6-sulfonic acid (ABTS), Potassium persulphate, Ascorbic acid, Ferrous sulphate (FeSO4), Hydrogen peroxide (H2O2), Sodium salicylate, Fetal bovine serum (FBS), Dimethyl sulfoxide (DMSO), Dulbecco in modified eagle medium (DMEM), 3-[4,5-Dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT), Phosphate buffered saline (PBS), Penicillin/Streptomycin antibiotic solution, Trypsin-Ethylene diamine tetra acetic acid (EDTA) was purchased from Gibco (USA), Ethidium bromide (ETBr) and Acridine Orange (AO) was purchased from Sigma Aldrich (USA). All other chemicals and reagents used in this study were of analytical grade. The experiments in this study were done using sterile distilled water.
4
In Vitro Evaluation of Antidiabetic Potential
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α-Amylase from porcine pancreas, α-glucosidase from Saccharomyces cerevisiae, para-nitrophenyl-glucopyranoside (pNPG), soluble starch, and 3-4,5-(dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were procured from SRL chemicals (Bangalore, India). Rat intestinal acetone powder (source of α-glucosidase), acarbose, Anti-GLUT2 antibody, anti-insulin antibody, 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA), acridine orange (AO), and ethidium bromide (EtBr) were from Sigma Chemical Co. (St. Louis, MO, USA). Glucose oxidase kit was purchased from AGAPE diagnostics (India). ELISA kits were purchased from Cytoglow Co. (India). Analytical and HPLC grade solvents were from E. Merck (India). The dried powder of Gymnema sylvestre was obtained from Nikhila Karnataka Central Ayurvedic Pharmacy (Mysore, India). Mouse pancreatic β-cell line (MIN6) was purchased from NCCS (Pune, India). Dulbecco's minimal essential medium (DMEM), Fetal Bovine Serum (FBS), and antimycotic solution were purchased from Himedia chemicals (India). All other chemicals used in the study were of analytical grade and purchased from Himedia chemicals (India).
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α-Amylase from porcine pancreas, α-glucosidase from Saccharomyces cerevisiae, para-nitrophenyl-glucopyranoside (pNPG), soluble starch, and 3-4,5-(dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were procured from SRL chemicals (Bangalore, India). Rat intestinal acetone powder (source of α-glucosidase), acarbose, Anti-GLUT2 antibody, anti-insulin antibody, 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA), acridine orange (AO), and ethidium bromide (EtBr) were from Sigma Chemical Co. (St. Louis, MO, USA). Glucose oxidase kit was purchased from AGAPE diagnostics (India). ELISA kits were purchased from Cytoglow Co. (India). Analytical and HPLC grade solvents were from E. Merck (India). The dried powder of Gymnema sylvestre was obtained from Nikhila Karnataka Central Ayurvedic Pharmacy (Mysore, India). Mouse pancreatic β-cell line (MIN6) was purchased from NCCS (Pune, India). Dulbecco's minimal essential medium (DMEM), Fetal Bovine Serum (FBS), and antimycotic solution were purchased from Himedia chemicals (India). All other chemicals used in the study were of analytical grade and purchased from Himedia chemicals (India).
5
Antibiotics and RNA isolation protocol
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All the antibiotics except penicillin G were procured from Sigma, while penicillin G was from Ranbaxy. Ethidium Bromide (EtBr) was from Sigma. Dipeptides were from Bachem. Qiagen RNAeasy kit was used for RNA isolation. DNase I amplification grade, Superscript III-RT, RNase out, Ethylene diamine tetraacetic acid (EDTA), Dithiotreitol (DTT), and random hexamers were from Invitrogen. 25 mM MgCl2 and 10 mM dNTP solutions were from Thermo Scientific. Eva green RT-qPCR master-mix from GBiosciences was used for RT-qPCR. Sodium-deoxy cholate and Phenol were from USB, Sodium cacodylate was from Fluka. Lysine monochloride, Ruthenium Red, and Glutaraldehyde were from Sigma. Pancreatic DNaseI and RNaseA are from Sigma. Proteinase K and Silver Nitrate are from Emresco. Nile Red was from Thermo fisher scientific.
6
Quantifying Ion Channel Expression
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The rCEnCs seeded with the density of 100 cells/mm2 films and TCP were cultured for 3 and 5 days. Trizol (Invitrogen, Carlsbad, CA, USA) and chloroform (Sigma-Aldrich, USA) were used to extract mRNA and centrifuged at 12,000 rpm in 4 °C for 15 min. The supernatant was transferred to a 1.5 mL Eppendorf tube. Iso-propanol (Sigma-Aldrich, USA) was added and kept in 4 °C overnight. Isolated mRNA was dissolved in RNase-DNase free water (Gibco, USA). The gene markers of voltage-dependent anion-selective channel 2 (VDAC2), voltage-dependent anion-selective channel 3 (VDAC3), chloride channel protein 2 (CLCN2), and sodium/bicarbonate co-transporter (NBC1) were evaluated and normalized using β-actin a housekeeping gene. Gene expression was measured by electrophoresis on 1% (w/v) agarose gel containing Ethidium Bromide (EtBr, Sigma-Aldrich, USA). Images were obtained under a UV light (FluorChem FC2, Alpha Innotech, San Leandro, USA) at 360 nm.
7
Assessing Cell Viability and Necrosis
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Viability and necrosis were performed as reported in earlier studies28 (link),86 (link),87 (link). Briefly, NHBE or A549 were seeded in 96-well cell culture plates and incubated for up to 6 h (10.000 cells/well), 24 h (10.000 cells/well), and 7 days (2.500 cells/well) with GO, FLG, or sFLG at increasing concentrations (0.05, 0.5, 5, 50, and 100 µg/mL). For FBS starvation tests, A549 cells were pre-cultured for 6 h or 24 h in a serum-free medium. Cells were then incubated with 10 μg/mL ethidium bromide (EtBr) (#46,067; Sigma-Aldrich) and 1 μM Calcein-AM (#C34852; Thermo Fisher). Viable cells, stained with green Calcein-AM, and necrotic cells, stained with red EtBr, were determined by fluorescence microscopy using a Cytation 5 Cell Imaging Multi-Mode Reader (20 × objective; BioTek) and analyzed with ImageJ 1.53. After image acquisition in living cells, samples were fixed and permeabilized in cold methanol for 4 min and then stained with 1 μg/mL Hoescht (#861,405; Sigma-Aldrich) to visualize DNA. Apoptosis was quantified by qualitative methods, as reported in earlier studies28 (link). Results are presented as the number of cells per field or as a percentage of necrotic or apoptotic cells vs. total (n = 3).
8
Rotavirus VP7 and VP4 Gene Detection
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RT-PCR was carried out for VP7 and VP4 gene fragments of 1062 and 876 bp respectively by using consensus primers (Beg9, End9 for VP7; VP41-17F, Con2 for VP4). The primer sequences used are Beg9 (5’GGC TTT AAA AGA GAG AAT TTC CGT CTG G3’), End9 (5’GGT CAC ATC ATA CAA TTC TAA TCT AAG3’). VP4_1-17F (5’GGC TAT AAA ATG GCT TCG C3’) and con2 (5’ATT TCG GAC CAT TTA TAA CC3’) [28 (link), 29 (link)]. The extracted RNA template was denatured for 2 min at 95 °C followed by reverse transcriptase PCR (RT-PCR) was carried out by using the Qiagen OneStep RT-PCR Kit (Qiagen/Westburg, The Nederland). The RT-PCR conditions involved initial reverse transcription at (30 min at 50 °C), polymerase activation at (95 °C for 15 min), 40 cycles of amplification (denaturation: 45 s at 94 °C; annealing (45 s at 45 °C for VP4 and 45 s at 50 °C for VP7), product extension (1 min at 72 °C) with final extension (10 min at 72 °C) [30 (link)]. The resulting PCR products were run on a polyacrylamide gel, stained with Ethidium Bromide (EtBr, Sigma Aldrich) and visualized under ultra violet light (UV-light).
9
Coenzyme Q Metabolism and Amyloid-beta
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Coenzyme Q2 and Q6 were purchased from Sigma Aldrich (#C8081, #C9504). Coenzyme Q9 was purchased from Cayman Chemical (#16866). Coenzyme Q10 was generously provided by Kaneka Corporation. Human Aβ25–35 (#AS-24227) and Human Aβ25–35 HiLyte™ Fluor 488-labeled (#AS-63308) were obtained from Anaspec. The fluorescent probes, Fluo-4 AM (#F23917), H2DCF-DA (#C6827), MitoSOX (#M36008), MitoTracker Red CMXRos (#M7512), Calcein-AM (#C34852), and Hoescht 33258 (#861405), were obtained from Thermo Fisher. Ethidium bromide (EtBr, #46067) was acquired from Sigma–Aldrich.
10
Caprine Slaughterhouse Derived G-SI Characterization
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Fresh G-SI was collected from a local caprine slaughterhouse of Saharanpur, India. Sodium chloride (NaCl), methanol, chloroform, ethanol, TX-100, chondroitin sulphate, potassium iodide (KI), hydrogen peroxide (H2O2), fluorescein diacetate (FDA) and ethidium bromide (EtBr) stains were purchased from Sigma-Aldrich (MO, USA). Phosphate buffer saline (PBS), ethylenediaminetetraacetic acid (EDTA), trypsin, sodium dodecyl sulphate (SDS), antibiotic solutions, Hematoxylin and Eosin (H&E) stain, collagenase enzyme, Dulbecco's modified Eagle medium (DMEM), DNA extraction kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit, alcian blue, 1, 9-dimethyl-methylene blue (DMMB), Mueller-Hinton (MH) agar, MH) broth, papain were purchased from Himedia (Mumbai, India). Double distilled and deionized water was used throughout the study.
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