DNA fiber assay was carried out as described previously (Castillo et al. 2014 (link)). Briefly, cells were pulse-labeled with 100 µM CldU and 250 µM IdU for 30 min. After labeling and treatment, cells were harvested, lysed, and spread on microscope slides. Immunofluorescence was carried out using α-IdU/α-BrdU (BD Biosciences) and α-CldU/α-BrdU (Abcam) and secondary antibodies, including Alexa fluor 488 (green) and Alexa fluor 555 (Invitrogen). Images were taken using a Nikon 80 microscope with a 60× lens and analyzed using ImageJ software. Statistics were calculated using Prism software.
α idu α brdu
Manufactured by BD
α-IdU/α-BrdU is a laboratory reagent used for detecting and quantifying DNA synthesis and cell proliferation in biological samples. It is a mixture of two antibodies that specifically recognize the incorporation of the thymidine analogs IdU (iododeoxyuridine) and BrdU (bromodeoxyuridine) into newly synthesized DNA. This reagent can be used in various experimental techniques, such as immunohistochemistry, flow cytometry, and microscopy, to identify and analyze proliferating cells.
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2 protocols using α idu α brdu
1
DNA Fiber Assay for Replication Dynamics
2
DNA Fiber Assay: Replication Dynamics
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DNA fiber assay was conducted as described previously (Castillo et al. 2014). Briefly, cells pretreated with JA2131 (10 µM) or DMSO for 2 h were labeled with 100 µM CldU for 30 min and exposed to 250 µM IdU (with or without 0.4 mM hydroxyurea treatment) for another 30 min or 3 h. After labeling and treatment, cells were lysed and DNA fibers stretched onto glass slides. Immunofluorescence was carried out using α-IdU/α-BrdU (BD Biosciences) and α-CldU/α-BrdU (Abcam) and secondary antibodies, including Alexa Fluor 488 (green) and Alexa Fluor 555 (Invitrogen). Images were taken using a Nikon 80 microscope and analyzed using ImageJ software. Statistics were calculated using Prism software. 5-Chloro-2’-deoxyuridine (CIdU) and 5-Iodo-2’-deoxyuridine (IdU) were from Sigma.
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