OptiMEM reduced serum medium and Lipofectamine 2000 reagent were purchased from Gibco Life Technologies (Grand Island, NY, USA). Protease inhibitor cocktail tablets (Complete Mini, EDTA-free) were purchased from Roche (Mannheim, Germany). Primary antibodies utilized were polyclonal rabbit anti-human TRPM2 antibody (Cat. #A300-414A, Bethyl Laboratories, Montgomery, TX, USA), polyclonal rabbit anti-human β-actin (Cat. #600-401-886, Rockland Immunochemicals, Limerick, PA, USA), polyclonal rabbit anti-human manganese superoxide dismutase (MnSOD) (Cat. #06-984, Millipore, Billerica, MA, USA), and monoclonal mouse anti-human Lamin B2 clone LN43 (Cat. #MA1-06104, Thermo Fisher Pierce, Pittsburgh, PA, USA). The secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated rabbit anti-mouse were purchased from Sigma (St. Louis, MO, USA). 2-Aminoethoxydiphenyl borate (2-APB), maintained as a 75 mM stock solution in dimethyl sulfoxide (DMSO), and 30% hydrogen peroxide solution were purchased from Sigma. The Fluo-4 NW Calcium Assay kit was purchased from Life Technologies. Comet Assay kit, which includes alkaline lysis solution, LMAgarose, 2-well CometSlides, SYBR Green, and EDTA, was purchased from Trevigen (Gaithersburg, MD, USA). CytoScan WST-1 cell proliferation assay was purchased from VWR International (Radnor, PA, USA).
Sybr green
Manufactured by Bio-Techne
Sourced in United States
SYBR Green is a fluorescent dye used in molecular biology applications for the detection and quantification of DNA. It binds to double-stranded DNA and emits a green fluorescent signal upon excitation, allowing for the real-time monitoring of DNA amplification during processes like quantitative PCR (qPCR).
Automatically generated - may contain errors
Lab products found in correlation
Comet assay kit, by Bio-Techne (5 mentions)
Lmagarose, by Bio-Techne (3 mentions)
Cometslide, by Bio-Techne (3 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
2 well cometslides, by Bio-Techne (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Ix51 fluorescence microscope, by Olympus (2 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (1 mentions)
2 aminoethoxydiphenyl borate 2 apb, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit, by Merck Group (1 mentions)
Cometassay, by Bio-Techne (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
R software, by R Project for Statistical Computing (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Low melting agarose, by Bio-Techne (1 mentions)
Prism software, by GraphPad (1 mentions)
Lsm 5 pascal inverted, by Zeiss (1 mentions)
Lysis solution, by Bio-Techne (1 mentions)
13 protocols using sybr green
1
Optimized Transfection and Cellular Assay
2
Comet Assay for DNA Damage Analysis
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Exponentially growing cells were cultured and treated with SDG (50 μM) at different time intervals prior to irradiation (2 Gy). Cells were processed for comet assay as per manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA). Briefly, cells (1 × 105 cells/mL in PBS) were mixed with LMAgarose® (1:10, v/v) and immediately pipetted onto CometSlide™. Cells were then lysed (4 °C, 30 min) and kept in dark for unwinding (RT). Electrophoresis was done in a horizontal electrophoresis unit at 18 volts (200 Amp) for 25 min. Slides were washed twice with distilled water, fixed in 70% ethanol and dried at 45 °C. DNA was stained by SYBR green (Trevigen). At least 150 cells were scored per group. Visual analysis of cells and comet tail length was measured using Comet Image Analysis software (Comet Assay IV, Perceptive Instruments Ltd., Haverhill, MA, USA). Images were captured on an Olympus IX51 fluorescence microscope using a monochrome CCD FireWire camera with a 40× objective lens.
3
DNA Damage Assessment by Comet Assay
4
Comet Assay for Asbestos-Induced DNA Damage
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Exponentially growing human pleural mesothelial cells were cultured in 6-well plates and exposed to 50 µM LGM2605 4 h prior to exposure to sterile crocidolite asbestos fibers (20 µg/cm2). As previously described [20 (link)], cells were processed for Comet assay as per manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA). Briefly, harvested mesothelial cells (1 × 105 cells/mL in 1× PBS) were mixed with LMAgarose® (1:10, v/v) and immediately pipetted onto CometSlides™. Cells were then lysed (4 °C for 30 min) and kept in the dark for unwinding (RT). Electrophoresis was done in a horizontal electrophoresis unit at 18 volts (200 Amp) for 25 min. Slides were washed twice with distilled water, fixed in 70% ethanol and dried at 45 °C. DNA was stained by SYBR green (Trevigen, Gaithersburg, MD, USA). At least 100 cells were scored per group. Visual analysis of cells and comet tail length was measured using Comet Image Analysis software (Comet assay IV, Perceptive Instruments Ltd., Haverhill, MA, USA). Images were captured on an Olympus IX51 fluorescence microscope using a monochrome CCD FireWire camera with a 40× objective lens.
5
TRPM2 Pathway Activation Analysis
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Opti-MEM reduced serum medium and Lipofectamine 2000 reagent were purchased from Invitrogen (Carlsbad, CA, USA). Protease inhibitor cocktail tablets (Complete Mini EDTA-free) were purchased from Roche (Mannheim, Germany). Primary antibodies utilized were poly-clonal rabbit anti-human TRPM2 antibody (cat. #A300-414A; Bethyl Laboratories, Montgomery, TX, USA), polyclonal rabbit anti-human β-actin (cat. #600-401-886), polyclonal rabbit anti-human apoptosis-inducing factor (AIF) (cat. #200-401-985) (both from Rockland Immunochemicals, Limerick, PA, USA) and monoclonal mouse anti-human poly(ADP-ribose) glycohydrolase (PARG) clone D8B10 (cat. #MABS61; Millipore). Two secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated rabbit anti-mouse were purchased from Jackson ImmunoResearch (West Grove, PA, USA). The CometAssay kit, which includes alkaline lysis solution, LMAgarose, 2-well CometSlides and SYBR-Green was purchased from Trevigen (Gaithersburg, MD, USA).
6
Comet Assay for DNA Double-Strand Breaks
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Formation of DNA DSBs was assayed by using the alkaline version of single-cell electrophoresis. Comet slides were stained by SYBR Green (Trevigen, Gaithersburg, MD, USA) and visualized by fluorescent microscopy (Olympus BX63, Tokyo, Japan). Comet assay parameters (TM and OTM) were evaluated by the ImageJ software. 50 nuclei were evaluated per treatment. Differences between control and treated cells in TM and OTM were analyzed by Kruskal–Wallis test followed by Dunn’s test with Benjamini–Hochberg adjustment in R software (R Foundation for Statistical Computing, Vienna, Austria; URL https://www.R-project.org/ ).
7
Comet Assay for Radiation and Hyperoxia
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Non-tumorigenic murine alveolar type II epithelial cells (C10) were challenged with hyperoxia and/or radiation and processed for comet assay analysis as per manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA). Briefly, cells (1 × 105 cells/mL in PBS) were mixed with LMAgarose® (1:10, v/v) and immediately pipetted onto Trevigen’s CometSlides™. Cells were then lysed (4 °C, 30 min) and kept in dark for unwinding at room temperature. Electrophoresis was done in a horizontal electrophoresis unit at 18 volts (300 mA) for 25 min. Slides were washed twice with deionized water, fixed in 70% ethanol and dried at 45 °C. DNA was stained by SYBR green (Trevigen). At least 150 cells were scored per group. Visual analysis of cells and comet tail length was measured using Comet Image Analysis software (Comet Assay IV, Perceptive Instruments Ltd., Haverhill, UK). Images were captured on an Olympus IX51 fluorescence microscope using a monochrome CCD FireWire camera (Perceptive Instruments Ltd., Haverhill, UK).
8
Comet Assay for DNA Damage Analysis
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Following UVB radiation, the cells were scraped and embedded in low melting point agarose on CometSlides™ (Trevigen, Gaithersburg, MD, USA) at 4°C for 10 min. The slides were then incubated with lysis buffer at 4°C for 1 h, followed by immersion in an alkaline unwinding solution for 30 min at room temperature (20–25°C). The slides were electrophoresed at 50 V, rinsed with distilled water and 70% ethanol, and then stained with SYBR®−Green (Trevigen) for 10 min. The DNA damage was visualized using a fluorescent microscope (IX71; Olympus Corporation, Tokyo Japan). These data were analyzed with analySIS LS Starter version 2.2 (Olympus Soft Imaging Solutions GmbH, Münster, Germany).
9
Comet Assay for DNA Damage Analysis
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The comet assay was performed using a comet assay kit (Trevigen, Gaithersburg, MD, USA). Cells were centrifuged, washed with ice-cold PBS, resuspended in a 0.5% (w/v) solution of low-temperature melting agarose in PBS at 37°C, and then layered onto comet slides (Trevigen). The agarose was incubated at 4°C for 30 min and then placed in a lysis solution containing 1% sodium lauryl sarcosinate, 10 mM Tris base, 2.5 mM NaCl, 100 mM EDTA, and 0.01% Triton X 100 (Trevigen) for 30 min at 4°C in the dark. The slide was subsequently immersed in alkaline unwinding solution containing 1 mM EDTA and 200 mM NaOH. Electrophoresis was performed in an alkaline buffer at 1 V/cm for 25 min at 4°C, and the gel was washed twice in distilled water. The washed slides were dried at 45°C for 20 min and then stained with SYBR Green (Trevigen). Images were obtained using a laser confocal scanning microscope (LSM 710; Zeiss, Heidenheim, Germany).
10
Comet Assay Reveals Allopurinol-AZA Toxicity
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The comet assay was used to identify the mechanisms behind the increased toxicity with allopurinol co-administration. This assay was performed according to the manufacturer’s instructions (Trevigen Inc., Gaithersburg, MD). HepaRG cells were seeded in 25-cm2 flasks and treated 24 h with allopurinol (100 μM), AZA (70 μM), AZA (70 μM) + allopurinol (100 μM) or untreated (control). After 24 h, cells were harvested and the cell suspension was mixed with 75 μL low melting agarose (Trevigen Inc.) in a density of 2 × 104 cells/mL and directly pipetted on agarose-precoated slides. Slides were stored at 4 °C for 30 min and subsequently submerged in lysis solution. After 60-min lysis, they were treated with alkaline unwending solution (pH > 13) for 60 min, followed by 30-min electrophoresis at 25 V. Slides were stained with SYBR green (Trevigen Inc.) and visualised and photographed by a digital camera (AxioCam MRm) attached to a fluorescent microscope (Axio imager.M1) using a 20× magnification.
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