The alkaline comet assay was performed as previously described (50 (link)), with some minor changes. Cells were plated in 6-well tissue culture-treated plates (Greiner) at 1.75 × 106 cells/well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 × 108 copies/well) or 50 μM etoposide (Sigma) for 20 h. Following infection, cells were then harvested, spun down, and resuspended in 0.5% low-melting-point agarose at 37°C. Samples then were spread onto agarose-coated slides (Cell Biolabs) and allowed to solidify for 20 min at 4°C. After agarose solidification, samples were incubated in lysis buffer (10 mM Tris-HCl, pH 10, 2.5 M NaCl, 0.1 M EDTA, 1% Triton X-100) for 1 h and then in the alkaline running buffer (0.3 M NaOH, 1 mM EDTA) for 30 min and finally electrophoresed at 300 mA for 30 min, all done at 4°C. Samples then were washed in double-distilled water (ddH2O) and fixed in 70% ethanol at 4°C. Cells were stained with Yoyo-1 (Life Technologies) for 15 min at room temperature and then washed with ddH2O and dried overnight. Images were acquired on the Zeiss Axio Imager Z1. Images were analyzed using the OpenComet plug-in for ImageJ.
6 well tissue culture treated plates
Manufactured by Greiner
6-well tissue culture-treated plates are a type of laboratory equipment designed for cell culture and biological experimentation. They provide a standardized and controlled environment for growing and maintaining cells in vitro. The plates have six individual wells, each with a flat, growth-promoting surface, allowing for the simultaneous cultivation of multiple cell samples or experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager z1, by Zeiss (6 mentions)
Yoyo 1, by Thermo Fisher Scientific (4 mentions)
Etoposide, by Merck Group (4 mentions)
Agarose coated slides, by Cell Biolabs (2 mentions)
Diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (2 mentions)
Anti 53bp1, by Cell Signaling Technology (2 mentions)
Anti rpa32, by GeneTex (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Hydroxyurea, by Merck Group (2 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (2 mentions)
Rabbit anti γh2ax, by Cell Signaling Technology (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Rabbit anti 53bp1, by Cell Signaling Technology (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Mouse anti egfp, by Takara Bio (1 mentions)
Rabbit anti actin, by Fortis Life Sciences (1 mentions)
Odyssey m, by LI COR (1 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
8 protocols using 6 well tissue culture treated plates
1
Alkaline Comet Assay for DNA Damage
2
Immunofluorescence Imaging of DNA Damage Markers
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U2OS cells were plated on glass coverslips in 6-well tissue culture-treated plates (Greiner Bio-one) overnight and then switched into plain or drugged media consisting of either 5.0 ng/mL PMA, 50.0 μM Etoposide, 5.0 μM NSC95397, or 10.0 μL DMSO for 24 hours. Cells were then fixed in 4% PFA for 15 minutes and permeabilized with 1% Triton-X 100 in PBS for 5–10 min at 4°C. Cells were then blocked with immunofluorescence (IF) block buffer (3% BSA in PBS) for 1 hour and incubated with primary antibodies: rabbit anti-γH2A.x (Cell Signaling 9718, 1:2000), rabbit anti-RPA32 (GeneTex GTX113004 1:2000), or rabbit anti-53BP1 (Cell Signaling S1778, 1:2000) overnight at 4°C. Coverslips were washed three times with PBS then incubated with secondary antibodies (Alexa Fluor 488 or 594) and Hoechst 33342 (1:2000) for 1 hour. Coverslips were then washed three times with PBS and mounted onto slides with ProLong™ Gold Antifade Mountant (Invitrogen) and sealed when dried. Images were obtained with a ZEISS LSM 980.
3
Western Blot Analysis of Protein Expression
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J-Lat 10.6 or 5A8 cells were plated in 6-well tissue culture-treated plates (Greiner Bio-one) at (5×106 cells/mL) in plain or drug media for 24 hrs. Cells were then lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris) for 15 min on ice and clarified by centrifugation at 14,800 x g for 15 minutes. Lysates were boiled in 4x sample buffer (250 mM Tris-HCL, 8% SDS, 0.2% Bromophenol Blue, 40% Glycerol, 20% β-mercaptoethanol) in preparation for SDS-PAGE using 12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed using mouse anti-eGFP (Takara, 1:2000), rabbit anti-Actin (Bethyl, 1:6000), mouse anti-gag (NIH-ARP#3537, 1:1000) followed by donkey anti-rabbit IgG 800CW (IRDye®,1:20000) or donkey anti-mouse IgG 680RD (IRDye®,1:20000) and imaged on a LI-COR Odyssey M.
4
Quantifying DNA Damage Response Markers
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Cells were plated in 6-well tissue culture-treated plates (Greiner) at 1.75 × 106 cells/well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 × 108 copies/well) or 50 μM etoposide (Sigma) for 20 h. For the EdU-IF experiments, EdU was added to the cells for 20 min. Cells were then permeabilized with 0.5% Triton X-100 in PBS at 4°C for 5 min and fixed in 4% PFA for 20 min. Samples were then washed in 1× PBS and incubated with blocking buffer (3% BSA, 0.05% Tween 20, and 0.04 NaN3 in PBS) for 30 min. Cells were probed with appropriate primary antibodies (anti-FLAG M2 [Sigma-Aldrich], anti-γH2AX, anti-RPA32 [GeneTex], or anti-53BP1 [Cell Signaling]) and then washed in PBST (0.05% Tween 20 in PBS) and probed with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Nuclei were stained with diamidino-2-phenylindole (DAPI; Life Technologies). Secondary staining for EdU was added as the last step and stained twice to ensure signal. Images were acquired on the Zeiss Axio Imager Z1, and mean fluorescence intensity (MFI) was analyzed using ImageJ.
5
DNA Combing Assay for Replication Dynamics
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The DNA combing assay was adapted from reference 52 (link). Cells were plated in 6-well tissue culture-treated plates (Greiner) at 1.75 × 106 cells/well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 × 108 copies/well) or 500 μM hydroxyurea (Sigma) for 20 h. Following infection, cells were incubated with 10 μM EdU (Invitrogen) for 20 min, harvested, spun down, and resuspended in 1× phosphate-buffered saline (PBS; Gibco). The cell suspension was added and lysed with lysis buffer (50 mM EDTA, 0.5% SDS, 200 mM Tris-HCl, pH 7.5) directly on a silane-coated slide (Electron Microscopy) and then incubated for 5 to 8 min. After incubation, the slide was tilted at a 45° angle to allow the droplet to roll down and then fixed with 3:1 methanol acetic acid for 15 min after the slide was completely dry. The slide then was washed with 1× PBS, blocked with 3% bovine serum albumin (BSA) for 30 min, and stained with secondary EdU mixture (Click-IT EdU imaging kit; Invitrogen) and DNA (Yoyo-1; Life Technologies). Microscopy was performed using the Zeiss Axio Imager Z1, and images were analyzed using ImageJ.
6
Quantifying DNA Damage Responses
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Cells were plated in 6-well tissue culture treated plates (Greiner) at 1.75 x 10 6 cell/ well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 x 10 8 copies/ well) or 50uM etoposide (Sigma) for 20 hrs. For the EdU-IF experiments, EdU was added to the cells for 20 minutes. Cells were then permeabilized with 0.5% Triton X-100 in PBS at 4˚C for 5 min, fixed in 4% PFA for 20 min. Samples were then washed in 1X PBS and incubated with blocking buffer (3% BSA, 0.05% Tween-20, and 0.04 NaN3 in PBS) for 30 minutes. Cells were probed with appropriate primary antibodies (anti-FLAG M2 [Sigma-Aldrich], anti-gH2AX, anti-RPA32 [GeneTex], or anti-53BP1 [Cell Signaling]), then washed in PBST (0.05% Tween-20 in PBS), and probed with Alexa-Fluor conjugated secondary antibodies (Life Technologies). Nuclei were stained with diamidino-2-phenylindole (DAPI; Life Technologies). Secondary staining for EdU was added as the last step and stained twice to ensure signal. Images were acquired on the Zeiss Axioimager Z1 and mean fluorescence intensity (MFI) was analyzed using ImageJ.
7
Quantifying DNA Damage by Alkaline Comet Assay
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Alkaline Comet Assay was performed as previously described (50) with some minor changes.
Cells were plated in 6-well tissue culture treated plates (Greiner) at 1.75 x 10 6 cell/ well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 x 10 8 copies/ well) or 50uM etoposide (Sigma) for 20 hrs. Following infection, cells were then harvested, spun down, and resuspended in 0.5% low melting point agarose at 37˚C. Samples were then spread onto agarose-coated slides (Cell Biolabs) and allowed to solidify for 20 min at 4˚C. After agarose solidification, samples were incubated in lysis buffer (10 mM Tris-HCl pH 10, 2.5M NaCl, 0.1M EDTA, 1% Triton X-100) for 1 hr, then in the alkaline running buffer (0.3M NaOH, 1 mM EDTA) for 30 min., and finally electrophoresed at 300mA for 30min -all done at 4˚C. Samples were then washed in ddH2O and fixed in 70% ethanol at 4˚C. Cells were stained with Yoyo-1 (Life Technologies) for 15 min at room temperature, then washed with ddH2O and dried overnight.
Images were acquired on the Zeiss Axioimager Z1. Images were analyzed using the OpenComet plug in for ImageJ.
Cells were plated in 6-well tissue culture treated plates (Greiner) at 1.75 x 10 6 cell/ well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 x 10 8 copies/ well) or 50uM etoposide (Sigma) for 20 hrs. Following infection, cells were then harvested, spun down, and resuspended in 0.5% low melting point agarose at 37˚C. Samples were then spread onto agarose-coated slides (Cell Biolabs) and allowed to solidify for 20 min at 4˚C. After agarose solidification, samples were incubated in lysis buffer (10 mM Tris-HCl pH 10, 2.5M NaCl, 0.1M EDTA, 1% Triton X-100) for 1 hr, then in the alkaline running buffer (0.3M NaOH, 1 mM EDTA) for 30 min., and finally electrophoresed at 300mA for 30min -all done at 4˚C. Samples were then washed in ddH2O and fixed in 70% ethanol at 4˚C. Cells were stained with Yoyo-1 (Life Technologies) for 15 min at room temperature, then washed with ddH2O and dried overnight.
Images were acquired on the Zeiss Axioimager Z1. Images were analyzed using the OpenComet plug in for ImageJ.
8
DNA Combing Assay for Replication Dynamics
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DNA combing assay was adapted from (52) . Cells were plated in 6-well tissue culture treated plates (Greiner) at 1.75 x 10 6 cell/ well and allowed to rest overnight. Cells were then infected with rAAV 2.5 at equal titers (1.4 x 10 8 copies/ well) or 500uM hydroxyurea (Sigma) for 20 hrs.
Following infection, cells were incubated with 10uM EdU (Invitrogen) for 20 min., then harvested, spun down, and resuspended in 1X PBS (Gibco). The cell suspension was added and lysed with lysis buffer (50mM EDTA, 0.5% SDS, 200mM Tris-HCl pH 7.5) directly on a silane-coated slide (Electron Microscopy), then incubated for 5 to 8 min. After incubation, the slide was tilted at a 45° angle to allow the droplet to roll down then fixed with 3:1 methanol acetic acid for 15 min after the slide was completely dry. Then the slide was washed with 1X PBS, blocked with 3% BSA for 30 min, and stained with secondary EdU mixture (Click-IT EdU imaging kit; Invitrogen) and DNA (Yoyo-1; Life Technologies). Microscopy was performed using the Zeiss Axioimager Z1 and images were analyzed using ImageJ.
Following infection, cells were incubated with 10uM EdU (Invitrogen) for 20 min., then harvested, spun down, and resuspended in 1X PBS (Gibco). The cell suspension was added and lysed with lysis buffer (50mM EDTA, 0.5% SDS, 200mM Tris-HCl pH 7.5) directly on a silane-coated slide (Electron Microscopy), then incubated for 5 to 8 min. After incubation, the slide was tilted at a 45° angle to allow the droplet to roll down then fixed with 3:1 methanol acetic acid for 15 min after the slide was completely dry. Then the slide was washed with 1X PBS, blocked with 3% BSA for 30 min, and stained with secondary EdU mixture (Click-IT EdU imaging kit; Invitrogen) and DNA (Yoyo-1; Life Technologies). Microscopy was performed using the Zeiss Axioimager Z1 and images were analyzed using ImageJ.
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