Adrian Salic, HMS assistant professor of cell biology, and Timothy Mitchison, HMS professor and deputy chairman of systems biology, have developed a chemical technique for detecting proliferation in cells and tissues that improves on commonly used methods. This tool will be useful to investigators wishing to evaluate the cell cycle to gain insight into cell metabolism, physiology, or pathology.
Their study, published in the Feb. 19 Proceedings of the National Academy of Sciences, involves the incorporation of a thymidine DNA precursor analogue, 5-ethynyl-2'deoxyuridine (EdU) into replicating DNA, followed by detection with a fluorescent azide using a copper-promoted chemical reaction.
Typically measuring proliferation in cells and tissues involves the incorporation of labeled deoxynucleosides into cellular DNA as it is replicating. This is followed by a detection method for quantifying or visualizing the newly synthesized DNA. The most frequently used labeled DNA precursors are tritiated thymidine, measured through audoradiography, and 5-bromo-2'deoxyuridine (BrdU), which utilizes an antibody detection system.
Each approach works well, but has limitations. Tritium is radioactive, making it cumbersome to work with. The detection method is labor intensive, and microscopic images have poor resolution. Though BrdU labeling is faster and allows for better imaging, it requires harsh denaturing agents for antibody detection that degrade the specimen. Furthermore, the size of the sample is limited by the ability of the antibody to penetrate fixed tissues, so the tissues must be sectioned. These restrictions are overcome by the use of EdU.
“It’s a method based on chemical detection rather than using an antibody, and it is more sensitive, much faster, and without some of the variables that BrdU and tritiated thymidine have,” explained Salic. “The unexpected advantage is that you can detect the EdU in tissues without harsh fixation and preparation conditions, such that the tissues you are going to look at will retain their structure and pristine form, unlike other methods, where the tissues take a beating during sample preparation.”
To test the chemical method, both live cells and mice were given EdU. Cells incorporated it into their DNA during replication, peppering the genetic material with this chemical group. Harvested cells and organs were reacted with a fluorescent azide that formed a covalent bond with EdU in the presence of a copper sulfate catalyst. When visualized under the microscope, the fluorescent azide-reacted EdU provided a stronger signal than the commonly used BrdU-labeled antibody method. And more of the EdU-containing molecules in the DNA could be detected. The technique also effectively visualized proliferating cells in the organs of the experimental mice.
“Because EdU is detected with a small chemical as opposed to a large antibody, it penetrates tissues rapidly and allows large chunks of tissue to be stained and examined under relatively low-power microscopes, giving an ensemble view of a tissue or organ that other methods cannot provide,” said Salic.
