Cell Cycle and Cell Proliferation Assays

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.

Nucleoside Analogs

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.

Assay Instrument Assay kits
EdU Microscope, flow cytometry, plate reader ab219801
BrdU Plate reader, microscope, flow cytometry ELISA: ab126556 / ab126572
IHC: ab125306
Antibody: ab6326

DNA-Staining Dyes

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.

Dye Instrument Note Assay kits
Propidium iodide Flow cytometer Ex/Em 536/617 nm ab14083, ab139418
Nuclear Green CCS1 Ex/Em 490/525 nm ab112116
Nuclear Red CCS1 Ex/Em 490/620 nm ab112117
DRAQ5TM Ex/Em 633 & 647/665–800 nm ab108410
DAPI Ex/Em 358/461  
Hoechst 33342 Ex/Em 350/461  
Hoechst 33258 Ex/Em 350/461  
7-AAD 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).

Assay Instrument Notes Assay kits
CFSE Flow cytometer Ex/Em 492/517 nm. Cytotoxic at higher concentrations. ab113853
CytoLabel Blue Flow cytometer, microscope Ex/Em 403/454 nm ab176726
CytoLabel Green Ex/Em 511/525 nm ab176735
CytoLabel Red Ex/Em 628/643 nm ab176736
CytoLabel Orange Ex/Em 542/556 nm ab176737

Protein Markers

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.

Clonogenicity Assays

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.

Senescence Assays

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.

Assay Instrument Assay kits
Beta-gal microscope, plate reader ab65351

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.

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Last updated: Apr 1, 2019 at 6:01 AM


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