The remaining cell suspension prepared as described for the MTS cell viability assay was used to perform the CometChip assay according to the manufacturer's alkaline assay protocol (Trevigen, Gaithersburg, MD) with some modifications. One hundred μL of the suspension were transferred to each of 5 wells of a 96‐well CometChip (Trevigen), with each well containing approximately 400 microwells. The cells were allowed to settle by gravity into the microwells for 30 min at room temperature. After cell loading, the CometChip was rinsed gently with 25 mL DPBS and then sealed with low melting point agarose (Trevigen). The cells then were treated with lysis solution (Trevigen) overnight at 4°C. DNA unwinding and electrophoresis were performed according to the Trevigen protocol. The CometChips then were neutralized in 0.4 M Tris–HCl buffer (pH 7.4, Sigma‐Aldrich) and equilibrated in 0.02 M Tris–HCl buffer (pH 7.4). Subsequently, the DNA was stained with 0.2 × SYBR® Gold (Invitrogen, Carlsbad, CA) overnight at 4°C and images were acquired using a BioTek Cytation 5 Image Reader (BioTek, Winooski, VT). Trevigen Comet Analysis Software was used to score the percentage of DNA in the comet tail (%DNA in Tail) for at least 300 cells from each culture insert.
Cometchip
Manufactured by Bio-Techne
The CometChip is a lab equipment product offered by Bio-Techne. It is designed to perform comet assays, a method used to measure DNA damage and repair in individual cells. The CometChip provides a standardized platform for conducting the comet assay, allowing for high-throughput analysis of DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
Lysis solution, by Bio-Techne (2 mentions)
Comet analysis software, by Bio-Techne (2 mentions)
Evos fl imaging system, by Thermo Fisher Scientific (2 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Biotek cytation 5 image reader, by Agilent Technologies (1 mentions)
Tris hcl buffer, by Merck Group (1 mentions)
Low melting point agarose, by Bio-Techne (1 mentions)
5 protocols using cometchip
1
CometChip Assay for DNA Damage
2
CometChip-Based DNA Damage Assay
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The comet assay was performed using a CometChip® (Trevigen) according to the manufacturer’s instructions. In brief, single-cell suspensions were prepared in 6 ml medium with 1.0 × 105 cells/ml density. Aliquots of 100 μl cells per well were applied to a CometChip and incubated in a tissue culture incubator for 10 min, with gentle shaking three times in 10 min intervals to spread cells evenly. Medium was removed and each CometChip from the 96-well CometChip® system was gently washed with 5 ml PBS twice. The CometChip was then covered with 6 ml of 1% 45°C low-melting agarose in PBS. After the solidification of the agarose, the slide was immersed in a lysis solution (Trevigen) overnight at 4°C. The CometChip was equilibrated twice in an alkaline solution at 4°C for 20 min, electrophoresed at 4°C for 50 min at 22 V in an alkaline solution, neutralized twice at 4°C for 15 min in fresh 0.4 M Tris (pH 7.4) buffer and then equilibrated at 4°C for 30 min in 20 mM Tris (pH 7.4) buffer. DNA in CometChips was stained with 0.2× SYBR® Gold in 20 mM Tris (pH 7.4) buffer at room temperature for 2 h. Images were acquired with a fluorescence microscope (BX53; Olympus) and the tail moment was calculated using the Comet analysis software (Trevigen).
3
CometChip-Based Alkaline Comet Assay
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Cells treated for 1 week with 10 µM of compound were plated onto a CometChip (Trevigen) at 2x105 cells/ml in a single cell suspension (Ge et al., 2014 (link)). Cells were incubated on the chip for 60 min at 37°C. The plate was then gently washed with PBS and coated with 1% low melting agarose. After the agarose was solidified by cooling at 4°C, the plate was washed with pre-chilled non-activated alkaline lysis buffer (2.5 M NaCl, 100nM Na2EDTA, 10mM Tris, pH 10). The chip was lysed overnight at 4°C by submerging the chip in the alkaline lysis buffer containing 1% Triton-X. The plate was then washed with PBS and placed in an electrophoresis chamber filled with pre-chilled alkaline electrophoresis buffer (2mM Na2 EDTA, 300 mM NaOH). Following a 40 min incubation at 4°C to allow for alkaline unwinding, electrophoresis was carried out at 80 volts for 30 min at 4°C. The chip was then neutralized with two incubations at 4°C in 400 mM Tris, pH 7.5 buffer, and equilibrated in 20 mM Tris, pH 7.4 at 4°C for 20 min. DNA staining was achieved by incubating the chip in 100 ml of 0.2X SYBR Gold overnight at 4°C. Fluorescent images were acquired on an Evos FL Imaging System (ThermoFisher).
4
Cell Cycle Synchronization and DNA Damage Repair Kinetics
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Cells were enriched at the G1 phase by incubating in growth medium supplemented with 1 μM (U2OS) or 2 μM (HAP1) palbociclib (hereafter referred to as the “working medium”) for 24 h prior to exposure to trabectedin or UV irradiation. XP2YO, and XP2OS were asynchronous prior to exposure to trabectedin or UV irradiation. Following this, cells were embedded in a 30 μm COMET chip (catalog no. 4250-096-01, Trevigen) and further incubated for 30 min at 37 °C in the working medium. The medium was removed, and cells were incubated in the working medium supplemented with 50 nM trabectedin for 2 h in the presence and absence of repair synthesis inhibitors (HAP1: 0.5 mM HU, 5 μM AraC, U2OS: 1 mM HU, 10 μM AraC, XP2YO and XP2OS: 4 mM HU, 40 μM AraC). After trabectedin treatment, cells were incubated in the working medium either with or without the repair synthesis inhibitors for varying repair periods. In the case of UV treatment, after removal of the medium, cells were subjected to irradiation using 5 J/m2 of UV-C (254 nm UV light). This was followed by incubation at 37 °C in the working medium, with or without the repair synthesis inhibitors, for different repair times. DNA strand breaks were examined utilizing the alkaline COMET chip assay.
5
Comet Assay Protocol for DNA Damage
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Cells treated for 1 week with 10 µM of compound were plated onto a CometChip (Trevigen) at 2x10 5 cells/mL in a single cell suspension 17 . Cells were incubated on the chip for 60 min at 37°C. The plate was then gently washed with PBS and coated with 1% low melting agarose. After the agarose was solidified by cooling at 4°C, the plate was washed with pre-chilled non-activated alkaline lysis buffer (2.5 M NaCl, 100nM Na2EDTA, 10mM Tris, pH 10). The chip was lysed overnight at 4°C by submerging the chip in the alkaline lysis buffer containing 1% Triton-X. The plate was then washed with PBS and placed in an electrophoresis chamber filled with pre-chilled alkaline electrophoresis buffer (2mM Na2 EDTA, 300 mM NaOH). Following a 40 min incubation at 4°C to allow for alkaline unwinding, electrophoresis was carried out at 80 volts for 30 min at 4°C. The chip was then neutralized with two incubations at 4°C in 400 mM Tris, pH 7.5 buffer, and equilibrated in 20 mM Tris, pH 7.4 at 4°C for 20 min. DNA staining was achieved by incubating the chip in 100 mL of 0.2X SYBR Gold overnight at 4°C. Fluorescent images were acquired on an Evos FL Imaging System (ThermoFisher).
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