Genotoxicity, also called DNA damage, was analyzed using an alkaline single-cell gel electrophoresis (comet) assay and a cytokinesis-block micronucleus (MN) assay. The procedures were performed according to previous studies [25 (link)]. The comet assay results revealed that the cells were mixed with the lysis solution on microscope slides coated with low-melting-point agarose. After electrophoresis, the slides were neutralized using a neutralization buffer and stained with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA). To quantify DNA damage, the tail moment and length were evaluated using the Comet v. 3 (Kinetic Imaging Ltd., Liverpool, UK). After the cells were incubated with cytochalasin B, an MN assay was used to assess rutin and TEGDMA pretreatment. Subsequently, after the cells were washed, they were resuspended in 75 mM KCl, fixed in a 3:1 mixture of methanol/acetic acid (Sigma-Aldrich, St. Louis, MO, USA), and stained with a 3% Giemsa solution. The MN assay was performed using a light microscope.
Methanol acetic acid
Manufactured by Merck Group
Sourced in Germany
Methanol acetic acid is a chemical compound used in various laboratory applications. It is a colorless, flammable liquid with a distinctive odor. The compound is commonly used as a solvent, reagent, and in the synthesis of other chemicals.
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Lab products found in correlation
Acetonitrile, by Merck Group (3 mentions)
Chlorophyll a, by Merck Group (2 mentions)
Epicatechin, by Extrasynthese (2 mentions)
Violaxanthin, by Merck Group (2 mentions)
Betulinic, by Merck Group (2 mentions)
Oleanolic and ursolic acid, by Merck Group (2 mentions)
Catechin, by Extrasynthese (2 mentions)
P coumaric acid, by Extrasynthese (2 mentions)
Quercetin 3 o glucoside, by Extrasynthese (2 mentions)
Quercetin 3 o rutinoside, by Extrasynthese (2 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid abts, by Merck Group (2 mentions)
Quercetin 3 o galactoside, by Extrasynthese (2 mentions)
Quercetin 3 o xyloside, by Extrasynthese (2 mentions)
Quercetin 3 o arabinoside, by Extrasynthese (2 mentions)
Gradient grade, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
Phloroglucinol, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Ethidium bromide, by Merck Group (1 mentions)
9 protocols using methanol acetic acid
1
Genotoxicity Assessment via Comet and MN Assays
2
Analytical Standards for Phytochemicals
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The analytical standards of chlorogenic acid, lutein, β-carotene, zeaxanthin, catechin, rutin, methanol acetic acid, ethanol, acetone, hexane, methanol, dimethylsulfoxide, butylated hydroxytoluene (BHT), sodium sulphate, Whatman filter paper number 1, isopropyl alcohol, N-hexane, potassium ferricyanide, 2,2-diphenyl-2-picrylhydrazyl (DPPH), potassium persulphate, ABTS, potassium dihydrogen orthophosphate, 5 mM p-nitrophenyl-α-d-glucopyranoside, sodium chloride, amylase enzyme, starch, acarbose, Folin–Ciocalteu reagent, sodium carbonate, ammonium hydroxide, acetic acid, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, hydrogen peroxide, acetone and hexane, sodium acetate, 2,4,6-Tris(2-pyridyl)-1,3,5-triazine (TPTZ), hydrochloric acid, ferric chloride and Trolox were purchased from Sigma Aldrich, Johannesburg, South Africa.
3
Karyotyping Chromosomes in Pluripotent Stem Cells
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Actively growing ES cells were treated with Demecolcin (20 μl of 10 μg/ml in 10 ml of media) for 1.5 hour. After detaching of cells with 1xPBS with Accumax (Millipore), cell suspension was incubated at 37°C in 0.56% KCl for 20 min. Cells were fixed with methanol: acetic acid 3:1 (Sigma), dropped onto glass slide and air dried. After the staining with Orcein (Sigma; one drop per slide), chromosomes were counted using microscope with phase contrast and 40x magnification. A total number of 20–40 good spreads were analyzed in four chiPS lines. For G-banding, slides with chromosomal spreads were incubated for 30 min at 80°C. After 5–7 sec digestion in 2% Trypsine (BD Difco) in Sorensen’s phosphate buffer (0.133 M Na2HPO4, 0.133 M KH2PO4, pH 6,8) chromosomes were stained for 2 min in Giemsa-Romanovski staining solution (5% Giemsa-Romanovski in Sorensen’s phosphate buffer). After the washing with distilled water and air-drying, 20–30 good mitotic spreads were captured using microscope under 100x magnification and karyotype was analysed using Lucia Karyo software (Lucia cytogenetics; http://www.lucia.cz ).
4
Cytogenetic Analysis of Cbx2 Null MEFs
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All animal experiments were conducted in accordance with the recommendations and guidelines of the Institutional Animal Care and Use Committee and the Public Health Service Policy on Humane Care and Use of Laboratory Animals of the National Institutes of Health. Cbx2 null and wild-type MEFs were collected at 18.5 d post coitum from timed male embryos of Cbx2 heterozygote matings (C.129P2-Cbx2tm1Cim/J; Coré et al., 1997 (link)) according to standard procedures. Primary cell cultures were maintained in high-glucose DMEM (Gibco/Thermo Fisher Scientific) supplemented with 10% FBS (Hyclone, GE Healthcare), 1/100 (vol/vol) sodium pyruvate, 1/100 (vol/vol) KClhCZIBPBHhf-iFeM4p/" target="_blank">L-glutamine, and 1/100 (vol/vol) penicillin/streptomycin (all Gibco/Thermo Fisher Scientific) before cytogenetic and molecular analysis of cultures at P2 and P5. Mitotic chromosome complements were spread onto glass slides following colchicine treatment (100 nM) for 2–5 h and hypotonic treatment with 75 mM KCl (Sigma-Aldrich) for 11 min before fixation with methanol/acetic acid (Sigma-Aldrich) or 2% PFA, 0.15% Triton X-100 for FISH and immunochemistry, respectively.
5
Comprehensive Phytochemical Analysis Protocol
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Acetonitrile, formic acid, methanol, all-trans-β-carotene, α-carotene, all-trans-lutein, neoxanthin, violaxanthin, antheraxanthin, β-cryptoxanthin, chlorophyll a, betulinic, oleanolic and ursolic acid, ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), methanol acetic acid, and phloroglucinol were purchased from Sigma-Aldrich (Steinheim, Germany). (−)-Epicatechin, (+)-catechin, procyanidin A2 and B2 chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, di-caffeic quinic acid, p-coumaric acid, 4-caffeoylquinic, kampferol-3-galactoside, quercetin-3-O-rutinoside, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-arabinoside, quercetin-3-O-xyloside, quercetin-deoxyhexo-hexoside caffeic acid, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-xyloside were purchased from Extrasynthese (Lyon, France). Acetonitrile for ultra-phase liquid chromatography (UPLC; Gradient grade) and ascorbic acid were purchased from Merck (Darmstadt, Germany).
6
Comprehensive Phytochemical Analysis Protocol
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Acetonitrile, formic acid, methanol, all-trans-β-carotene, all-trans-lutein, all-trans-zeaxanthin, violaxanthin, chlorophyll a, chlorophyll b, chlorophyllide b, pheophytin a, pheophytin b, betulinic, oleanolic and ursolic acid, ABTS 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), methanol acetic acid, and phloroglucinol were purchased from Sigma-Aldrich (Steinheim, Germany). (-)-Epicatechin, (+)-catechin, procyanidin B2,, caffeic acid, p-coumaric acid, 3-O-caffeoylquinic, 5-O-caffeoylquinic, ferulic acid, galloyl glucose, caftaric acid, luteolin 7-O-galactoside, apigenin 7-O-glucoside, kaempferol-3-O-galactoside, quercetin-3-Orutinoside, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-arabinoside, quercetin-3-O-xyloside, cis-piceid, trans-piceid, cis-resveratrol were purchased from Extrasynthese (Lyon, France). Acetonitrile for ultra-phase liquid chromatography (UPLC; Gradient grade) and ascorbic acid were purchased from Merck (Darmstadt, Germany).
7
Metaphase Karyotyping Analysis
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Karyotype analysis was performed after cells were arrested at metaphase by incubation with Colcemid (Invitrogen Corporation, Grand Island, NY, USA), maintained in a hypotonic solution (0.075 M KCl), fixed with methanol/acetic acid 3:1 (Merck, Milan, Italy), and stained with Giemsa using standard laboratory protocols. At least 20 metaphases were analyzed using MackType software (Nikon Corporation, Tokyo, Japan) according to the International System for Human Cytogenetic Nomenclature.
8
DNA Fiber Analysis for Replication Asymmetry
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DNA fiber analyses were carried out as described previously [33 (link)]. For measuring sister fork asymmetry, cells were labeled with CldU (33 μM) for 30 min followed by exposure to 2 mM HU for 2 h and chased with IdU (340 μM) for 40 min before harvesting in PBS. Cells were lysed (lysis buffer: 200 mM Tris-HCl (pH 7.4), 50 mM EDTA, 0.5% SDS) and DNA fibers stretched onto glass slides and fixed in methanol:acetic acid (3:1, Merck). After rehydration in PBS, these were denatured with 2.5 M HCl for 1 h, washed with PBS and blocked with 2% BSA in PBS containing 0.1% Tween 20 for 30 min. The newly replicated CldU and IdU tracks were immunostained using anti-BrdU primary and appropriate secondary antibodies. Coverslips were mounted using Antifade Gold (Invitrogen). Images were acquired on a Leica DMI 6000 fluorescence microscope and analyzed using Fiji software [34 (link)]. Statistics were calculated in Graph Pad Prism (GraphPad Software Inc., San Diego, CA, USA).
9
Cytokinesis-Block Micronucleus Assay
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We performed the cytokinesis-block micronucleus assay to measure the numbers of cells that contained micronuclei. After the last medium transfer, the QU- DB bystander cells received 0.8 µg/ml of cytochalasin B. The MRC5 bystander cells received 2 µg/ml of cytochalasin B. QU-DB flasks that contained cytochalasin B were allowed to incubate for 45 hours, whereas MRC5 flasks were allowed to incubate for 24 hours (1.5 doubling time). Following incubation, the culture medium was removed, the cells in the flasks were washed with PBS, air-dried, and subsequently fixed. MRC5 cells were fixed once with absolute methanol and QU-DB cells with a combination of methanol:acetic acid (Merck, Germany) at a 3:1 ratio for three times. After drying, cells were stained with 10% giemsa for 5-6 minutes and viewed at ×400 magnification. For accuracy, one examiner scored the slides twice. Each time the number of cells that contained MNBN were counted (4 (link)).
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