Studying mitotic chromosome structure using flow cytometry
Every organism is built from cells which are created through cell division. Genetic information, a manual encoding of how cells are built and maintained, is stored in the form of genomic DNA. Genomic DNA forms rod-shaped mitotic chromosomes during cell division which is necessary for the accurate transfer of genomic information. If genomic information is damaged or lost, the daughter cells could die or become harmful to the organism (e.g. give rise to cancer).
Dr Kumiko Samejima, a postdoc from the Earnshaw lab at the University of Edinburgh, has been investigating how these mitotic chromosomes are formed using vertebrate cells as a model system. Previous studies showed that protein complexes called condensins are essential to forming rods. Kumiko has modified cell lines so that the condensin proteins can be rapidly depleted upon the addition of a chemical. She has further genetically modified these cells so they enter mitosis synchronously.
With these tools in place, she has been able to analyse the cell population entering mitosis minute-by-minute using inter-disciplinary approaches including high-resolution microscopy, genomic and proteomic analysis, and polymer modelling. Her data demonstrated that condensin organises DNA into loops arranged like a helical staircase along the axis of the cylinder. This work led to a high-profile publication in Science (2018) which has already been cited over 400 times.
Flow cytometry analysis and cell sorting have been integral to these studies since obtaining a cell population uniformly depleted of target protein (e.g. condensin) is critical to generating reliable experimental data. Kumiko used flow cytometry to measure the levels of each fluorescently-labelled target protein in individual cells. This is essential to establish and maintain cell lines that exhibit maximal and even levels of depletion of target proteins. Cell sorting is also crucial to eliminate those cells with high levels of target proteins that have managed to evade the depletion procedure.
Kumiko has also collaborated with Facility Manager, Martin Waterfall, to establish a new protocol to synchronise cells. In 2019 they jointly obtained a SULSA technology seed project grant to develop a flow cytometry-based method to obtain cells in the later stages of mitosis termed anaphase/telophase. Once established, this will become a valuable tool for understanding how genomic DNA is transferred safely into new cells.
Flow cytometry is a laser-based technology that allows the multi-parameter measurement of characteristics of biological particles in a single cell suspension as they flow in a liquid medium past an excitation light source. This can be applied to whole cells as well as prepared cellular components such as nuclei or organelles. The underlying principle is that light is scattered, and fluorescence is emitted, as light from the excitation source strikes individual moving particles.
Flow cytometry is particularly important for biological research because it allows qualitative and quantitative examination of whole cells and cellular constituents that have been labelled with a wide range of commercially available reagents, such as dyes, reporter proteins and monoclonal antibodies.
We have 20 parameter multiplexing capabilities with aseptic cell sorting technologies, including 4-way simultaneously sorting and single-cell cloning. The facility also offers HTS plate acquisition with support for experimental design and analysis.
Benchtop analysers available for multiparameter analysis:
Also available is a state-of-the-art high-speed cell sorter:
The FACSAria cell sorter provides a method for dissecting a heterogeneous mixture of biological cells into discreet sub-populations, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument which provides fast, objective and quantitative recordings of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
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