The chemicals used were 100 mM stock solution of BrdU (Merck, B5002) and 10 mM stock solutions of PARG inhibitor (PDD0017273, Tocris, 5952; Merck, SML1781), PARP inhibitors (KU0058948, Axon Medchem, Axon 2001; Olaparib, ApexBio, A4154), FEN1 inhibitor (UOS-33991; compound 17 in ref. 52 (link) and synthesized in-house as described in ref. 11 (link)), REV1 TLS inhibitor (JH-RE-06, Axon Medchem, Axon 3002) and EdU (Cambridge Bioscience, CAY20518) were prepared in dimethyl sulfoxide (DMSO). CldU (Merck, C6891) and IdU (Merck, I7125) were dissolved directly in culture medium at a final concentration of 2.5 mM, and thymidine (Merck, T1895) in culture medium at 200 mM. 1 mCi per ml 3H-Thymidine (PerkinElmer, NET027W005MC) in 2% ethanol and 99% MMS were added directly to culture medium to a final concentration of 2 µCi per ml and 0.01%, respectively.
I7125
Manufactured by Merck Group
Sourced in United States
The I7125 is a laboratory equipment product from Merck Group. It is a compact and versatile device designed for various scientific applications. The core function of the I7125 is to provide accurate and reliable measurements or processing of samples in a laboratory setting. Detailed specifications and intended use are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
C6891, by Merck Group (19 mentions)
B5002, by Merck Group (5 mentions)
Ab6326, by Abcam (4 mentions)
Prism 6, by GraphPad (3 mentions)
Pdd0017273, by Bio-Techne (1 mentions)
Ku0058948, by Axon Medchem (1 mentions)
Ibitreat slide 6 0, by Ibidi (1 mentions)
Axio imager fluorescence microscope, by Zeiss (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
H8627, by Merck Group (1 mentions)
Sml0569, by Merck Group (1 mentions)
M1404, by Merck Group (1 mentions)
Axio imager m2 fluorescence microscope, by Zeiss (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Clone b44, by BD (1 mentions)
A10044, by Thermo Fisher Scientific (1 mentions)
B9285, by Merck Group (1 mentions)
Orca flash4.0 v2, by Hamamatsu Photonics (1 mentions)
Las x software, by Leica (1 mentions)
25 protocols using i7125
1
DNA Replication Inhibitors Screening
2
Fluorescent Nucleoside Tracking Assay
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3
DNA Fiber Assay for Replication Dynamics
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DNA fiber assay was performed as previously described [25 (link)] with the following modification: 1.5 × 105 A2780 and A2780Cis cells were seeded into 6-well plates. The following day cells were synchronized at the G1/S boundary by 6 µM aphidicolin for 24 h [26 (link)]. Cells were washed 2 times with warm PBS, one time with warm RPMI media, and treated as indicated. During the last hour of drug treatment, cells were labeled with CldU 25 μM (Sigma-Aldrich, C6891) for 30 min and then with 250 μM IdU (Sigma-Aldrich, #I7125) for 30 min. Cells were harvested by trypsinization, resuspended in PBS, and samples were diluted to approximately 7 × 105 cells/mL. Fiber spreading, denaturation, fixation, and staining were performed in ibiTreat µ-slide VI 0.4 (Ibidi, #80606). Fluorescence images were captured using a NikonTi2 fluorescence microscope with the 40× objective (with MilliQ water) with excitation wavelengths of 488 and 568 nm, and analyzed using the ImageJ software. At least 100 unidirectional forks labeled with both CldU and IdU were measured for every condition using Fiji [27 (link),28 (link)]. Antibodies used are listed in Table S1 .
4
DNA Replication Dynamics Analysis
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DNA fiber labeling analysis was used to assess DNA replication fork progression. 48 hours after siRNA transfection, HeLa cells were labeled for 20 minutes with 25 μM IdU (I7125, Sigma, St Louis, MO) followed by 20 minutes labeling with 250 μM CldU (C6891, Sigma, St Louis, MO). Cells were harvested and suspended in ice cold PBS. Next 2 μl of the cell suspension was deposited over the slide and 10 μl of lysis buffer (0.5% SDS, 200 mM Tris-HCl pH 7.4, 50 mM EDTA) was added. The slides were tilted to 15° to stretch the DNA fibers. Slides were then air dried, fixed in 3:1 methanol: acetic acid, denatured in 2.5M HCl and blocked with 5% BSA in PBS. Then slides were incubated with rat anti-CldU and mouse anti-IdU for one hour followed by goat anti-mouse 568 and goat anti-rat Alexa Fluor 488. Fork velocity and stalled replication fork (only red) were measured and statistical analysis was performed using Prism 6 (GraphPad Software).
For fork recovery, HeLa cells were labeled for 100 minutes with 25 μM IdU and the last 60 minutes was added with 1 μM CPT. Both the IdU and CPT were washed, then followed by 20 minutes labeling with 250 μM CldU. Cells were then processed for fiber combing as explained before. Recovered forks (red followed by green) and unrecovered forks (only red) were counted for 300 replication structures and their percentage was calculated 52 (link)
For fork recovery, HeLa cells were labeled for 100 minutes with 25 μM IdU and the last 60 minutes was added with 1 μM CPT. Both the IdU and CPT were washed, then followed by 20 minutes labeling with 250 μM CldU. Cells were then processed for fiber combing as explained before. Recovered forks (red followed by green) and unrecovered forks (only red) were counted for 300 replication structures and their percentage was calculated 52 (link)
5
Measuring DNA Replication Fork Speed
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To measure fork speed, cells were incubated with 20 μM 5-chlorodeoxyuridine (CldU; C6891, Sigma-Aldrich) for 20 minutes, followed by 20 minutes of incubation with 100 μM 5-iododeoxyuridine (IdU; I7125, Sigma-Aldrich). Genomic DNA purification and combing on glass coverslips were performed using materials and a protocol from Genomic Vision. DNA fiber analysis was performed as previously described (82 (link)). The DNA was stained with anti-CldU antibody (ab6326, Abcam) and anti-IdU antibody (347580, BD Biosciences) for 1 hour, followed by incubation with secondary antibodies for 45 minutes. Images were acquired by Nikon Eclipse E800 epifluorescence microscope using a ×40 objective and analyzed with ImageJ software (NIH).
6
BrdU and IdU Labeling in Zebrafish
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Stage 41 juveniles were placed in a 0,4 mg/ml BrdU or IdU solution (B5002 and I7125 respectively, Sigma) in ERM for 16 hr and rinsed in abundant fresh ERM before transferring to a tank. Adult fish were placed in a in a 0.4 mg/ml BrdU or IdU solution in fish water for 24 or 48 hr, and washed extensively before returning them to the tank.
7
DNA Fiber Assay for Replication Dynamics
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A DNA fiber assay was performed as previously described (40 (link)). To detect fork progression, cells were incubated consecutively with 50 μM IdU (I7125, Sigma) and 250 μM CldU (C6891, Sigma) for 20 min. To determine fork integrity, cells were either incubated with 50 μM IdU followed by 4 mM HU and 250 μM CldU or consecutively labeled with IdU and CldU, followed by HU treatment. After IdU and/or CldU labeling, cells were harvested and DNA fibers were spread on microscopy slides, followed by fixation with 3:1 methanol:acetic acid and denaturation with 2.5 M HCl. Slides were then incubated with a primary antibody and then a secondary antibody. Fiber images were obtained using a Zeiss Axio Imager fluorescence microscope. Fiber lengths were measured manually using the Zeiss Zen Pro software. At least 150 fibers were quantified for each sample.
8
DNA Replication Dynamics Analysis
9
Labeling Dividing Cells for TBI Assessment
10
DNA Fiber Assay for BCL6 Regulation
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The DNA fiber assay was performed as previously described (58 (link)). Cells were plated at 50% confluence onto 15 cm plates and allowed to adhere overnight. The cells were treated with 10 μM COMP7 and BCL6 siRNAs alone or in combination with pcDNA3.1-BCL6 for 48 hours. The treated cells were subsequently incubated with 5-iodo-2′-deoxyuridine (IdU; I7125, Sigma) and 5-chloro-2′-deoxyuridine (CIdU; C6891, Sigma). After labeling, 2.5 μL of the cell suspension (approximately 2,500 cells) was spotted onto glass slides, followed by the addition of 7.5 μL lysis buffer. CIdU and ldU were detected by confocal microscopy using red (ab6326, Abcam) and green (347580, BD Biosciences) fluorescent secondary antibodies, respectively. The ratio of green and red fiber track lengths (IdU/CIdU) was quantified using the ImageJ software.
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