Cell synchronization is a process by which cells in a culture at different stages of the cell cycle are brought to the same phase. Cell synchrony is a vital process in the study of cells progressing through the cell cycle as it allows population-wide data to be collected rather than relying solely on single-cell experiments. The types of synchronization are broadly categorized into two groups; physical fractionization and chemical blockade.
Physical Separation
Physical fractionation is a process by which continuously dividing cells are separated into phase-enriched populations based on characteristics such as the following:
Cell density
Cell size
The presence of cell surface epitopes marked by antibodies
Light scatter
Fluorescent emission by labeled cells.
Given that cells take on varying morphologies and surface markers throughout the cell cycle, these traits can be used to separate by phase. There are two commonly used methods.
Centrifugal Elutriation
(Previously called: counter streaming centrifugation) Centrifugal elutriation can be used to separate cells in different phases of the cell cycle based on their size and sedimentation velocity (related to sedimentation coefficient). Because of the consistent growth patterns throughout the cell cycle, centrifugal elutriation can separate cells into G1, S, G2, and M phases by increasing size (and increasing sedimentation coefficients) with diminished resolution between G2 and M phases due to cellular heterogeneity and lack of a distinct size change.
Larger cells sediment faster, so a cell in G2, which has experienced more growth time, will sediment faster than a cell in G1 and can therefore be fractionated out. Cells grown in suspension tend to be easier to elutriate given that they do not adhere to one another and have rounded, uniform shapes. However, some types of adherent cells can be treated with trypsin and resuspended for elutriation as they will assume a more rounded shape in suspension.
Flow Cytometry and Cell Sorting
Flow cytometry allows for detection, counting, and measurement of the physical and chemical properties of cells. Cells are suspended in fluid and put through the flow cytometer. Cells are sent one at a time through a laser beam and the light scatter is measured by a detector. Cells or their components can be labeled with fluorescent markers so that they emit different wavelengths of light in response to the laser, allowing for additional data collection.
For quantitative cell cycle analysis, cells are usually fixed with ethanol and stained with DNA-binding dyes like propidium iodide, Hoechst 33342, DAPI, 7-Aminoactinomysin D, Mithramycin, DRAQ5, or TO-PRO-3, allowing for determination of phase by DNA quantity. However, if these cells have been fixed, they are dead and cannot be maintained for continued growth. Cells can also be resuspended in media and dyed with non-toxic dyes to maintain living cultures. Cells can also be genome edited such that some cellular proteins are made with conjugated fluorescent tags such as GFP, mCherry, and Luciferase that can be used to detect and quantify those components. For example, chimeric histone H2B-GFP constructs can be made and used to measure DNA content and determine replication status as a means of discerning cell phase. Light scatter measurements can be used to determine characteristics like size, allowing for distinction of cell phase without tagging.
Flow cytometers can be used to collect multiparameter cytometry data, but cannot be used to separate or purify cells. Fluorescence-activated cell sorting (FACS) is a technique for sorting out the cells based on the differences that can be detected by light scatter (e.g. cell size) or fluorescence emission (by penetrated DNA, RNA, proteins or antigens). The system works much like flow cytometry, but will also charge each cell droplet after it has been measured based on a defined parameter. The charged droplet will then encounter an electrostatic deflection system that will sort the cell to a different container based on that charge. This allows cells to be separated on the basis of fluorescent content or scatter.
To summarize, flow cytometry alone can be used to gather quantitative data about cell cycle phase distribution, but flow cytometry in coordination with FACS can be used to gather quantitative data and separate cells by phase for further study. Limitations include: