Techniques to Study Cell Cycle

• • Flow cytometry is used to identify which phase of cell cycle a population of cells are in. This is because it gives information about the DNA contents in the cells, especially when DNA in cells are fluorescently labelled, as DNA contents are different in G1, S & G2/M phase.
• • Flow cytometry operates by: cells being harvested & dispersed into a single cell, where DNA is labelled using propidium iodide (PI); running the single cells through a flow cytometer in a 'single file' between laser beam & photon detectors; laser lights getting scattered when cells 'flow' past, where the amount of scatter (or brightness of fluorescence of each cell if labelled) can be read by optical detectors to give information about size & complexity.
• • BrdU labelling, a substitute for thymidine, can be used to label cells in S phase by adding immunohistochemical marks which are incorporated into DNA as it replicates. This is because the cells that have immunofluorescent labelling have BrdU contained in the nuclei, so were cells that were in S phase when BrdU was added.
• • BrdU labelling operates through: pulse labelling (typically 1 hr), which involves brief administration of BrdU or EdU & the longer these markers are present, the more cells will stain; & pulse chase, where normal thymidine are added at various times after administration of BrdU to compete with it, allowing the collection/tracking of where cells go after proliferation at various times after administration.
• • Post-translational histone modifications (pHH3) can be used to label cells of any stage in the M phase, as it is based on identifying H3 extensive phosphorylation upon the Ser-residues.
• • Post-translational histone modifications operate by: using antibodies that specifically recognises the phosphorylated form of histone H3, such as Ki-67 that detects nuclear protein associated with cell proliferation to identify proliferating cells.
• • Western blotting can be used to identify which cyclins are expressed in a population of cells, as it is a method that separates & identifies proteins. It is used to find expression levels of proteins but does not allow knowledge of where these proteins are located.
• • Western blotting operates by: proteins being crushed & immunohistochemically labelled (if needed); proteins are separated according to size & charge by electrophoresis; and results are 'blotted' onto a synthetic membrane, where the blot would be at the location with appropriate molecular weight of the protein present.
• • The cyclins for different phases of cell cycle are: cyclin D (D1, D2, D3) for G1-Cdk; cyclin E for G1/S-Cdk; cyclin A for S-Cdk; & cyclin B for M-Cdk.