Dual DNA Replication Labeling Protocol

Cells were labeled with 100 μM iododeoxyuridine (IdU) for 10 min, then with 100 μM chlorodeoxyuridine (CldU) for 20 min. DNA fibers were spread as described previously^{26 (link)} and stained with primary antibodies (mouse anti-BrdU/IdU from BD Bioscience; rat anti-BrdU/CldU from Accurate Chemical) and fluorescence-conjugated secondary antibodies (Alexa Fluor 488-anti-rat and Texas Red-conjugated anti-mouse from Invitrogen). Fibers were imaged using Zeiss Axio Imager M2 and measured using AxioVision software (SE64 version 4.9.1).

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Kim J., Hu Z., Cai L., Li K., Choi E., Faubert B., Bezwada D., Rodriguez-Canales J., Villalobos P., Lin Y.F., Ni M., Huffman K.E., Girard L., Byers L.A., Unsal-Kacmaz K., Peña C.G., Heymach J.V., Wauters E., Vansteenkiste J., Castrillon D.H., Chen B.P., Wistuba I., Lambrechts D., Xu J., Minna J.D, & DeBerardinis R.J. (2017). CPS1 maintains pyrimidine pools and DNA synthesis in KRAS/LKB1-mutant lung cancer cells. Nature, 546(7656), 168-172.

Publication 2017

mouse anti-BrdU/IdU Alexa Fluor 488-anti-rat Texas Red-conjugated anti-mouse Axio Imager M2

Alexa fluor 488 Antibodies Brdu Cells Fluorescence antibodies Iododeoxyuridine Mouse

Corresponding Organization : The University of Texas Southwestern Medical Center

Other organizations : Children's Medical Center, The University of Texas MD Anderson Cancer Center, Pfizer (United States), KU Leuven

Top 5 similar protocols

Variable analysis

independent variables

Exposure time of cells to iododeoxyuridine (IdU) (10 min)
Exposure time of cells to chlorodeoxyuridine (CldU) (20 min)

dependent variables

DNA fiber length
Fluorescence intensity of stained DNA fibers

control variables

Concentration of IdU (100 μM)
Concentration of CldU (100 μM)
DNA fiber spreading method
Primary antibodies (mouse anti-BrdU/IdU, rat anti-BrdU/CldU)
Fluorescence-conjugated secondary antibodies (Alexa Fluor 488-anti-rat, Texas Red-conjugated anti-mouse)
Imaging method (Zeiss Axio Imager M2)
Measurement software (AxioVision software (SE64 version 4.9.1))

controls

Positive control: Not specified
Negative control: Not specified

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!