This article outlines the options available to you if you require a cell cycle or cell proliferation assay.
These assays have three functions: monitoring the growth rate of a cell population, detecting daughter cells in a growing population, and analyzing the proportions of cells during different stages of the cell cycle.
Some of these methods are listed below:
- Nucleoside analogs’ (for example, BrdU and EdU) incorporation during DNA synthesis
- DNA staining dyes’ utilization for cell cycle analysis
- Dye dilution assays
- Protein markers including PCNA, Ki67 and MCM-2
- Clonogenicity assays
- Senescence assays
Information about these assay types is listed below. Some of the most popular assay kits are EdU, propidium iodide and CFSE.
An alternative option is to analyze cell proliferation using cell viability assays which measure the rate of cellular metabolism. Examples of these assays include MTT, MTS, and resazurin among others. Cellular esterase cleaved dyes, mitochondrial membrane potential dependent dyes, ATP and ADP assays, and assays measuring glycolytic flux and oxygen consumption can also be used.
During DNA replication, two assays - bromodeoxyuridine (BrdU) and ethynyldeoxyuridine (EdU) – detect the incorporation of BrdU or EdU into newly synthesized DNA. While it is easy to directly label EdU -either by using a fluorescent dye, biotin for colorimetric, or fluorometric detection via streptavidin-HRP – BrDU must be detected using antibodies. The BrdU protocol is therefore harsher than EdU staining, which is consistent with further antibody staining.
||Microscope, flow cytometry, plate reader
||Plate reader, microscope, flow cytometry
||ELISA: ab126556 / ab126572
Often used in flow cytometry, DNA-staining dyes measure cell populations’ DNA content and assay for cell cycle state. The dye most frequently used is propidium iodide.
||Ex/Em 536/617 nm
|Nuclear Green CCS1
||Ex/Em 490/525 nm
|Nuclear Red CCS1
||Ex/Em 490/620 nm
||Ex/Em 633 & 647/665–800 nm
||Ex/Em 488/647 nm
Dye Dilution Assays
Cells retain the dyes in dye dilution assays over multiple generations. Half of the parent cells’ dye is received by daughter cells, and a flow cytometer is used to analyze assays. The dye which has been established the longest is carboxyfluorescein succinimidyl ester (CFSE).
||Ex/Em 492/517 nm. Cytotoxic at higher concentrations.
||Flow cytometer, microscope
||Ex/Em 403/454 nm
||Ex/Em 511/525 nm
||Ex/Em 628/643 nm
||Ex/Em 542/556 nm
For the analysis of cell proliferation within tissue samples, or often within cell culture, it is common to use antibodies to stain for the presence of Ki67, PCNA, or MCM-2.
Clonogenic/clonogenicity assay is the standard technique for assaying cell proliferation, although it is not often used at high throughput. This assay plates out cells at a low density. Subsequently, it counts the number of colonies that are formed.
The overexpression and accumulation of the endogenous lysosomal beta-galactosidase (SA-beta-gal) is the most common marker of senescent cells. A colorimetric or fluorometric substrate is used to detect Beta-gal activity.
||microscope, plate reader
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