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These assays monitor the growth rate of a cell population, detect daughter cells in a growing population, or analyze the proportions of cells in different stages of the cell cycle.
Alternatively, cell proliferation can also be analyzed with cell viability assays that measure the rate of cellular metabolism, such as MTT, MTS, resazurin and similar assays, mitochondrial membrane potential dependent dyes, cellular esterase cleaved dyes, ATP and ADP assays, and assays that measure glycolytic flux and oxygen consumption.
Bromodeoxyuridine (BrdU) and ethynyldeoxyuridine (EdU) assays measure the incorporation of BrdU or EdU into newly synthesized DNA during DNA replication. Unlike BrdU, which is detected using antibodies, EdU can be easily directly labeled, either with a fluorescent dye or biotin for colorimetric or fluorometric detection via streptavidin-HRP. Edu staining is consistent with further antibody staining, unlike the harsher BrdU protocol.
Microscope, flow cytometry, plate reader
Plate reader, microscope, flow cytometry
DNA-staining dyes are commonly used in flow cytometry to measure the DNA content in cell populations and assay for cell cycle state. Propidium iodide is the mostly commonly used dye.
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
The dyes in dye dilution assays are retained within cells over multiple generations. Daughter cells receive half of the dye of parent cells and assays are analyzed on a flow cytometer. Carboxyfluorescein succinimidyl ester (CFSE) is the longest established dye.
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 sometimes within cell culture, it is common to use antibodies to stain for the presence of Ki67, PCNA, or MCM-2.
Although little used at high throughput, the classical method of assaying cell proliferation is to use a clonogenic/clonogenicity assay. In this assay, cells are plated out at a low density and then the number of colonies formed is counted.
The most common marker of senescent cells is the overexpression and accumulation of the endogenous lysosomal beta-galactosidase (SA-beta-gal). Beta-gal activity is detected using a colorimetric or fluorometric substrate.
microscope, plate reader
Learn more about:
Cell viability assays based on cell metabolism/enzyme activity
Measure the rate of continuing cellular activities, such as metabolism.
Cytotoxicity assays based on cytolysis/membrane leakage
Test for cell membrane damage, either by measuring the leakage of cellular enzymes or staining with membrane-impermeable dyes.
Apoptosis assays/cell death analysis
Measure the markers present in different types of cell death.