U2OS cells were exposed to IR (10 Gy) and processed according to manufacturer's recommendations (Trevigen). Cells were trypsinized at the indicated time points and resuspended at 105 cells/ml in PBS. Cells were combined with low melting agarose at 1:10 ratio and spread over the CometSlide. Slides were allowed to dry at 4°C for 10 min, then immersed in lysis buffer (Trevigen) overnight. The next day, the slides were immersed neutral electrophoresis buffer (two 15‐min washes) followed by electrophoresis at 31 V for 45 min. Subsequently, the slides were incubated for 30 min in DNA precipitation solution followed by 30 min in 70% ethanol. Slides were dried and stained with SYBR Gold (Invitrogen). Images were taken using the EVOS FL Cell Imaging System microscope, and the tail moment was quantified using the CaspLab software. For each condition, at least 50 cells were analyzed.
Lysis buffer
Manufactured by Bio-Techne
Lysis buffer is a solution used to break down and lyse cells, releasing their contents for further analysis. It is a key component in various biological research and diagnostic applications, enabling the extraction and isolation of cellular components such as proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Lmagarose, by Bio-Techne (4 mentions)
Cometslide, by Bio-Techne (4 mentions)
Sybr gold, by Thermo Fisher Scientific (4 mentions)
Ix71 microscope, by Olympus (3 mentions)
Sybr green, by Roche (3 mentions)
Mini sub cell gt electrophoresis tray, by Bio-Rad (2 mentions)
Cygreen, by Enzo Life Sciences (2 mentions)
Alkaline cometassay kit, by Bio-Techne (2 mentions)
Comet assay kit, by Bio-Techne (2 mentions)
L name, by Bio-Techne (1 mentions)
Sybr green 1, by Merck Group (1 mentions)
Cometassay reagent kit, by Bio-Techne (1 mentions)
Apotome fluorescence microscope, by Zeiss (1 mentions)
Low melting agarose, by Merck Group (1 mentions)
Te2000 e, by Nikon (1 mentions)
Sybr green, by Merck Group (1 mentions)
Dm6 b upright, by Leica (1 mentions)
Dfc360 fx camera, by Leica (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Dm6000b upright microscope, by Leica (1 mentions)
15 protocols using lysis buffer
1
Quantifying DNA Damage in U2OS Cells
2
Modulation of ROS/RNS in Breast Cancer Cells
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MDA-MB-231, MCF-7 and HCC38 cells were plated at a density of 5 × 104 per well and incubated for 24 h. In order to understand the time course during which ROS/RNS generation occurred, cells were exposed to cortisol (1 μM) and NE (1 μM) for 15, 30, and 90 minutes. Control wells were left untreated. Following this period, the medium was removed and cells lysed using 500 μl lysis buffer (Trevigen). Lysates were then collected and ROS/RNS levels were quantified using multiple-step amperometry using a stainless steel counter electrode and non-leak Ag|AgCl reference electrode. Measurements of the current were obtained at +0.3 V, +0.45 V, +0.62 V and +0.85 V for a duration of 30 s.
Additional measurements were also carried out in MDA-MB-231 and MCF-7 cells to understand how ROS/RNS levels were altered when the cells were incubated with RU486 (1 μM) (GR antagonist), propranolol (1 μM) (β-adrenergic receptor antagonist) 1400 W dihydrochloride (10 μM) (iNOS inhibitor; Tocris, UK), L-NAME (100 μM) (non-specific NOS inhibitor; Tocris, UK) and PP2 (10 μM) (Src inhibitor; Abcam, UK) for 30 minutes prior to hormone treatment.
Additional measurements were also carried out in MDA-MB-231 and MCF-7 cells to understand how ROS/RNS levels were altered when the cells were incubated with RU486 (1 μM) (GR antagonist), propranolol (1 μM) (β-adrenergic receptor antagonist) 1400 W dihydrochloride (10 μM) (iNOS inhibitor; Tocris, UK), L-NAME (100 μM) (non-specific NOS inhibitor; Tocris, UK) and PP2 (10 μM) (Src inhibitor; Abcam, UK) for 30 minutes prior to hormone treatment.
3
Neutral Comet Assay for DNA Double-Strand Breaks
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A neutral comet assay was used to specifically assess the amount of DNA DSBs in cells treated with Veliparib, CPT, or both. In individual wells of a six-well plate, cells were incubated with 0.4 mL of Trypsin for 10 minutes. 1 mL of medium was added to the trypsin and the cells centrifuged at 1000 g for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 500 μL of PBS. The suspended cells were added to prewarmed low-melting agarose at 37°C (10 μL of cells to 90 μL of agarose). The mix was pipetted onto a CometSlide (4250, Trevigen) and spread equally across the slide before being allowed to set for 30 minutes at 4°C. Following this, the slides were submerged in cold Lysis Buffer (4250, Trevigen) for 30 minutes on a shaker, and then in cold Tris/Borate/EDTA buffer. Electrophoresis was run for 15 minutes at 21V in a Mini-Sub Cell GT electrophoresis tray (Bio-Rad). Subsequently, cells were fixed in 70% ethanol and allowed to dry overnight. DNA was stained with Cygreen (GEN-105, ENZO) for 30 minutes, and slides were imaged on an EVOS fluorescence microscope (AMG) with appropriate filters for GFP imaging. Images acquired were processed using Open Comet software and the olive moment for each group of cells was calculated [83 (link)].
4
Alkaline Comet Assay for DNA Damage
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Upon cell harvest, cells were rinsed in cold phosphate-buffered saline (PBS), and 1 × 105 cells/mL was suspended in PBS prior to the comet assay as described in the Alkaline Comet Assay kit protocol (Trevigen Inc., Gaithersburg, MD, USA). The cell suspension was combined with low-melting-point agarose, and the samples were transferred with a pipette onto comet slides prior to storage in ice-cold lysis buffer (Trevigen Inc.) at 4 °C for 35 min. The comet slides were electrophoresed with alkaline electrophoresis buffer and treated with 70% ethanol for 6 min before drying. DNA was stained with SYBR green I (Sigma) and photographed under a confocal microscope.
5
Neutral COMET Assay Protocol
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The neutral COMET assays were performed
using a COMET assay reagent kit (Trevigen) according to the manufacturer’s
protocol. MDA-MB-231 cells were treated as indicated for 30 h, and
then collected and washed with ice-cold PBS. The washed cells were
mixed with 1% low-melting agarose (Sigma) and spread on the slides
(Trevigen). The slides were incubated with lysis buffer (Trevigen)
overnight at 4 °C. After electrophoresis in neutral running buffer,
the slides were stained with SYBR gold (Life Technologies) and visualized
by an ApoTome fluorescence microscope (Zeiss). The images were analyzed
with CASPlab software,58 (link) and the percentage
of DNA in the tail and tail moment were used to compare different
samples.
using a COMET assay reagent kit (Trevigen) according to the manufacturer’s
protocol. MDA-MB-231 cells were treated as indicated for 30 h, and
then collected and washed with ice-cold PBS. The washed cells were
mixed with 1% low-melting agarose (Sigma) and spread on the slides
(Trevigen). The slides were incubated with lysis buffer (Trevigen)
overnight at 4 °C. After electrophoresis in neutral running buffer,
the slides were stained with SYBR gold (Life Technologies) and visualized
by an ApoTome fluorescence microscope (Zeiss). The images were analyzed
with CASPlab software,58 (link) and the percentage
of DNA in the tail and tail moment were used to compare different
samples.
6
Quantifying DNA Double-Strand Breaks via Neutral Comet Assay
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To assess the amount of DNA DSBs generated by siRNA treatments, a neutral comet assay was used. Cells were cultured in a six-well plate and treated as outlined above. For the comet assay, these cells were then incubated with 0.4 mL of Trypsin for 10 min. One milliliter of medium was added so that the cells could be suspended and then centrifuged at 1000 × g for 5 min. The cells were then resuspended in 500 μL of fresh PBS and added to prewarmed low-melting point agarose at 37 °C (10 μL of cells to 90 μL of agarose). This suspension was pipetted onto a CometSlide (4250, Trevigen) and spread equally before being allowed to set for 30 min at 4 °C. The slides were then submerged in cold Lysis Buffer (4250, Trevigen) for 30 min on a shaker and then in cold Tris/Borate/EDTA buffer. Electrophoresis was run for 15 min at 21 V in a Mini-Sub Cell GT electrophoresis tray (Bio-Rad). Subsequently, cells were fixed in 70% ethanol and allowed to dry overnight. DNA was stained with Cygreen (GEN-105, ENZO) for 30 min, and slides were imaged on an EVOS fluorescence microscope (AMG) with appropriate filters for green fluorescent protein imaging. Images acquired were processed using the Open Comet software and the olive moment for each group of cells was calculated98 (link).
7
Comet Assay for DNA Damage
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Using ice-cold phosphate-buffered saline (PBS), cells were washed and suspended at a concentration of 1 × 105 cells ml−1. The comet assay was performed by using the Alkaline Comet Assay kit (Trevigen Inc., Gaithersburg, MD, USA) as per the manufacturer’s instructions. Briefly, the cell suspension was mixed with low melting point-agarose, chilled, and stored in ice-cold lysis buffer (Trevigen Inc.) at 4 °C. The comet slides were immersed sequentially in alkaline electrophoresis buffer and 70 % ethanol. Comet cell DNA was then stained with SYBR Green (Sigma-Aldrich), and cells were imaged by using a confocal microscope (TE-2000-E; Nikon, Tokyo, Japan).
8
Comet Assay for DNA Damage Detection
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U2OS FIT were induced for 7 d with doxycycline 1 µg/ml and treated for 1 h with 50 nM CPT and washout (3 h). Cells were collected and resuspended in cold PBS. 2 × 104 cells were mixed with 0.8% wt/vol low melting point, previously equilibrated to 37°C, and then spread onto a comet slide (Trevigen; CometAssay Kit, catalog number 4250–050-ESK). Slides were incubated for 20 min at 4°C to allow solidification of the low melting point. Cells were lysed overnight in lysis buffer (Trevigen). Slides were incubated in cold electrophoresis buffer (300 mM sodium acetate and 100 mM Tris, pH 8.3) for 1 h at 4°C and then subjected to electrophoresis for 30 min at 21 V/300 mA. Samples were rinsed twice in water, fixed in 70% ethanol for 20 min at 4°C, and then dried at 37°C. DNA was stained with SYBR Gold (Thermo Fisher Scientific). Microscopy was performed on a Leica DM6 B upright digital research microscope equipped with a DFC360 FX Leica camera at 10× magnification. The images were analyzed using the Open Comet plugin (http://www.cometbio.org ) for Fiji.
9
Comet Assay for Cellular DNA Damage
10
Neutral Comet Assay for DNA Damage
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Cells were treated with siRNA 48 h prior to IR (3 Gy, Mainance Millenium Sample Irradiator containing a Cs-137 sealed source) and harvested at indicated timepoints. Neutral comet assays were carried out according to manufacturer's guidelines (Trevigen). Briefly, cells were harvested and combined with LMAgarose (Trevigen) at a final concentration of 1 × 105 cells/ml and loaded onto polylysine slides. The agarose plugs were allowed to solidify at 4°C for 1 h before being immersed in lysis buffer (Trevigen) overnight at 4°C. Slides were then equilibrated in cold electrophoresis buffer (100 mM Tris pH9.0, 300 mM NaAz) for 30 min prior to electrophoresis for 1 h at 24 V. The DNA was precipitated for 30 min at RT in DNA precipitation buffer (1 M NH4Ac in EtOH) and washed with 70% EtOH for a further 30 min. Slides were allowed to dry overnight at 37°C prior to staining with SYBR green (Roche, S7563). Images were acquired on a DeltaVision integrated microscope system using the Applied Precision SoftWoRx acquisition software mounted on an IX71 Olympus microscope with a 10× air objective (Imsol). All images were taken as single slices using a CoolSNAP HQ2 ICX-285 CCD camera. Comet analysis was performed using the CometScore software from Tritek Corporation.
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