For intracellular FACS staining of γH2AX, 1 × 106 cells were washed twice with PBS and fixed with 3% paraformaldehyde for 10 min at 37°C. Cells were then permeabilised with 90% methanol for a minimum of 30 min at 4°C or left overnight at −20°C. After fixation and permeabilisation cells were washed three times with 0.5% BSA in PBS and were blocked with 5% mouse serum (inactivated) in PBS and stained with 5 μl γH2AX Alexa Fluor 488 antibody for 1 hrs at RT in the dark. Cells were washed once again and co‐stained with 25 μg ml−1 propidium iodide containing 100 ng μl−1 RNase A for 20 min at RT in the dark. Subsequent fluorescence intensities were examined by flow cytometry (FACSCanto II). A minimum of 1 × 104 cells per sample were analysed. The comet assay was carried out with the OxiSelectTM Comet Assay Kit (Catalog Number STA‐350, Cell Biolabs) (Biocat, Heidelberg, Germany).
Oxiselect comet assay kit
Manufactured by Cell Biolabs
Sourced in United States, Germany
The OxiSelect Comet Assay Kit is a laboratory tool used to measure DNA damage in individual cells. It provides a simple and sensitive method for the detection of DNA strand breaks, alkali-labile sites, and other types of DNA damage. The kit includes all necessary reagents and components to perform the comet assay procedure.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (9 mentions)
Axiovert 200m, by Zeiss (3 mentions)
Ix71 fluorescence microscope, by Olympus (2 mentions)
Vista green dna dye, by Cell Biolabs (2 mentions)
Etoposide, by Merck Group (2 mentions)
Vista green dna dye, by Olympus (2 mentions)
Xm10 digital camera, by Olympus (2 mentions)
Metavue imaging, by Molecular Devices (2 mentions)
X cite 120 fluorescence illumination system, by Nikon (2 mentions)
Eclipse te2000 u microscope, by Nikon (2 mentions)
Eclipse 80i microscope, by Nikon (2 mentions)
Evos microscope, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Apotome, by Zeiss (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Other antibodies, by Cell Signaling Technology (1 mentions)
Cd31 antibody, by R&D Systems (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
106 protocols using oxiselect comet assay kit
1
Intracellular FACS Analysis of γH2AX
2
Comet Assay for Nanoemulsion-Induced DNA Damage
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ARPE-19 and HMC3 cells were plated at a density of 50,000 cells/well, and grown to confluence in 24-well plates for 7 days. Twelve hours before the experiment, the cells were treated with different nanoemulsions (at 20%). After the treatment with nanoemulsions, the isolation of cells was carried out, followed by the preparation of a single-cell suspension to embed the cells in agarose gel. After proceeding the lysis, the cells were submitted to alkaline treatment followed by horizontal electrophoresis (30 min, 35 volts, 300 mA), as described by Doktorovova et al. [27 (link)]. After neutralization, the samples were stained with Vista Green DNA Dye and visualized in Inverted Fluorescence Microscope/Coupling Stage, Zeiss Axiovert 200M (Carl Zeiss, Germany) using the 20× objective. The assay was performed using the OxiSelectTM Comet Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). The results were analyzed using the OpenComet software (www.cometbio.org , accessed on 15 February 2021); the percentage of DNA in the tail and the olive tail moment (OTM) were used to determine the extent of DNA damage.
3
Alkaline Comet Assay Protocol
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The comet assay was performed under alkaline conditions using the OxiSelectTM Comet assay kit (Cell Biolabs Inc. San Diego, CA, USA) following the manufacturer’s instructions. Briefly, electrophoresis was carried out on 1 × 105 cells layered on comet slides for 30 min at 25 V and 300 mA. Then the slides were stained with 100 μL/well of diluted Vista Green DNA Dye (Cell Biolabs Inc.). The comet images were captured with an Olympus IX71 fluorescence microscope (200X magnification).
4
Alkaline Comet Assay for DNA Damage
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The comet assay was performed under alkaline conditions using the OxiSelectTM Comet assay kit (Cell Biolabs Inc. San Diego, CA, USA) following the manufacturer′s instructions. Briefly, electrophoresis was carried out on 1 × 105 cells layered on comet slides for 30 min at 25 V and 300 mA. Then the slides were stained with 100 μL/well of diluted Vista Green DNA Dye (Cell Biolabs Inc.). The comet images were captured with an Olympus IX71 fluorescence microscope (200X magnification). The distance of DNA migration from each cell was measured from the body of the nuclear core to the trailing edge of the comet. The comet lengths of 50 individual cells were measured for each treatment group.
5
Comet Assay for Radiation-Induced DNA Damage
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For the comet assay, HCT116 cells were transfected with 100 nmol of a mixture of p73i-1 and -2 or with 0.5 μg CMV-p73, followed by γ-irradiation. At 48 hpi, the irradiated cells were fixed in 70% ethanol and subjected to cell dissociation in ice-cold phosphate-buffered saline (PBS) containing 20 mM EDTA (without Mg2+ and Ca2+). The assay was performed using a OxiSelectTM comet assay kit (Cell Biolabs Inc., San Diego, CA, USA) according to the manufacturer’s recommendations. Embedded cells were treated with lysis buffer at pH 7 (i.e. non-alkaline) to release the double-stranded DNA. For data processing, each comet picture was measured using ImageJ software, version 1.45 (National Institutes of Health, Bethesda, MD, USA), and the extent of damage in individual cells was calculated as described previously [3 (link)].
6
Isolation and Characterization of Ailanthone
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Ailanthone (purity ≥ 98%) was extracted and isolated from Ailanthus altissima as reported previously, and its chemical structure was identified by spectral data (Supplementary Figure )15 (link). The reagents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), propidium iodide (PI), ribonuclease A (RNase A) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were supplied by Sigma (St. Louis, MO, USA). The TACSTM Annexin V-FITC/PI staining assay kit was obtained from Trevigen Inc. (Gaithersburg, MD, USA). The OxiSelectTM comet assay kit was purchased from Cell Biolabs (San Diego, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin (PS) were purchased from Gibco BRL (Life Technologies, NY, USA). Protease inhibitor cocktail tablets were purchased from Roche Applied Science (Mannheim, Germany). The CD31 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Other antibodies were purchased from Cell Signaling Technology, Inc. (Manchester, NH, USA).
7
Comet Assay for DNA Damage Evaluation
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The OxiSelectTM Comet Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) was used to evaluate DNA damage with or without drug treatment. Briefly, cells (1 × 105 cells/mL) were mixed with molten agarose (Cell Biolabs, Inc.) at 37 °C at a ratio of 1:10 (v/v), and then transferred to comet slides and incubated in the dark for 15 min. Slides were immersed in pre-chilled lysis buffer for 1 h and then with freshly prepared pre-chilled Alkaline Solution (pH > 13) for 30 min at 4 °C in the dark. Slides were electrophoresed in alkaline buffer at 1 volt/cm for 30 min. The cells were stained with 1x Vista Green DNA Dye for 15 min at RT and then viewed with a 4× fluorescence microscope. The percentage of DNA in the tail were analyzed with Casplab_1.2.3b2 (CaspLab Comet Assay Software, CASPLab, http://casplab.com/ ) [30 (link)]. At least 50 randomly selected cells were analyzed per sample.
8
Comet Assay for DNA Damage Evaluation
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The intensity of DNA damage was evaluated using the OxiselectTM Comet assay kit purchased from Cell Biolabs, Inc (San Diego, CA, USA). The alkaline version of the test was used for its more sensitivity than the neutral version and was performed as described by the manufacturer. Ethidium bromide-stained nucleoids were visualized by epifluorescence microscope (Olympus BX51) using FITC filter (excitation: 460–500 nm, emission 510–560 nm). Cellular nuclei were photographed using the CoolSnap HQ digital camera. The intensity of DNA damage was determined as described previously by Park et al.50 (link). 100 comets/slide were scored visually and classified according to the tail intensity and assigned a value of 0, 1, 2, 3, or 4 (0 indicates undamaged, 1 indicates slightly damaged, 2 indicates moderately damaged, 3 indicates severely damaged, and 4 indicates very severely damaged). The total score for 100 comets was again divided by factor of 5 to yield an arbitrary value ranging from 0 (if the counted a hundred comets are all undamaged) to 80 (if the counted 100 comets are all very severely damaged).
9
Comet Assay for Chondrocyte DNA Damage
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Chondrocytes (mLncZFHX2 fl/fl or C57BL/6 mice) were transfected with adenovirus or siRNA and cultured under hypoxic conditions for 1 week. The comet assay was performed using an OxiSelectTM Comet Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, heat the Comet Agarose bottle at 90–95 °C in a water bath and transfer the bottle to a 37 °C water bath. The cell samples were combined with Comet Agarose in a 1:10 ratio (v/v), mixed well by pipetting, and immediately transferred onto the top of the Comet Agarose Base Layer (75 μL per well). After solidification, immerse the slides in the Lysis Buffer for 30 min at 4 °C in the dark. The slides were then transferred to a pre-chilled tris-borate-EDTA (TBE) buffer for 10 min. A voltage was applied to the chamber for 10–20 min at 1 V/cm in the TBE buffer. The slides were then immersed in pre-chilled double distilled H2O for 2 min and repeated twice more. After replacing the slides with cold 70% ethanol for 5 min, the slides were stained with Vista Green DNA Dye for 15 min. Fluorescence images were acquired using a fluorescence microscope (BX51TRF; Olympus, Tokyo, Japan) and analyzed using the CaspLab software.
10
Comet Assay for CPC Genotoxicity
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The OxiSelect™ Comet Assay Kit (Cell Biolabs, INC, San Diego, CA, USA) was used to verify whether CPC is genotoxic on MCF-7 cells. 21 Briefly, after an overnight drug exposure to 0, 5, or 100 µM CPC or 100 µM etoposide (Sigma-Aldrich Corporation, St. Louis, MO, USA) employed as positive control, MCF-7 were collected and washed with PBS. DNA was stained with DAPI (1 µL/mL, final concentration; 100 µL/well). The slides were observed with an EVOS microscope (Thermo Fisher Scientific, CA, USA) using a DAPI filter.
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