Cell Proliferation Assays

Kamalendra Yadav, National Agri‐Food Biotechnology Institute, Punjab, India
Nitin Singhal, National Agri‐Food Biotechnology Institute, Punjab, India
Vikas Rishi, National Agri‐Food Biotechnology Institute, Punjab, India
Hariom Yadav, National Agri‐Food Biotechnology Institute, Punjab, India

Published online: August 2014

DOI: 10.1002/9780470015902.a0002566

Abstract

Cells are structural and functional unit of our body that control and maintain the function of all unicellular and multicellular organisms. The process of cell proliferation and differentiation plays a vital role from the time of embryogenesis to development of whole organism from single‐ or double‐cell embryo and continues its crucial role in maintenance of adult tissue homoeostasis by recycling the old cells with new cells. In addition, abnormal cell proliferation is also associated with various human diseases like cancer. Therefore, cell proliferation assays become crucial to scrutinise the rate of cell proliferation in both in vitro and in vivo conditions. These assays follow different parameters to determine the rate of cell proliferation like rate of deoxyribonucleic acid synthesis, metabolic activity of cells, different antigens associated with cell proliferation and adenosine triphosphate concentration. In the present article, authors have tried to summarise different cell proliferation assays commonly used in laboratories to determine cell proliferation.

Key Concepts:

  • Cell proliferation plays a vital role in regular tissue and cellular homoeostasis for proper growth, development and maintenance of organism.

  • Cell proliferation assays are mainly designed based on three concepts, that is, (1) measuring rate of DNA replication, (2) analysis of metabolic activity and (3) cell surface antigen recognitions.

  • Rate of DNA replication can be analysed by using radioactive or labelled nucleotide analogues, that is, 3H‐thymidine‐ and BrdU‐based assays.

  • Metabolic activity‐based assays include MTT, XTT, WST, resazurin and ATP measurements.

  • Cell proliferation antigen‐based assay targets antigens present in proliferating cells such as markers like Ki‐67, topoisomerase IIB, phosphohistone H3 and PCNA.

Keywords: cell proliferation; cell assays; metabolic activity; ATP; BrdU; MTT assay; rate of DNA synthesis

Figure 1.

Scheme of labelled nucleotide analogue incorporation during DNA replication to measure cell proliferation rate.
Figure 2.

Cell proliferation assay using 3H‐thymidine nucleotide incorporation and radioactive measurements.
Figure 3.

Schematic representation of BrdU incorporation assay into the cell culture system.
Figure 4.

Brief scheme of metabolic activity measurement using MTT and other tetrazolium salts to access cell proliferation.
Figure 5.

Scheme of antigen‐based cell proliferation assay.
close

References

Ahmed SA, Gogal RM and Walsh JE (1994) A new rapid and simple nonradioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assays. Journal of Immunological Methods 170: 211–224.

Al‐Nasiry S, Geusens N, Hanssens M, Luyten C and Pijnefnborg R (2006) The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Human Reproduction 22: 1304–1309.

Anoopkumar‐Dukie S, Carey JB, Conere T et al. (2005) Resazurin assay of radiation response in cultured cells. British Journal of Radiology 78: 945–947.

Barltrop J and Owen T (1991) 5‐(3‐carboxymethoxyphenyl)‐2‐(4,5‐dimethylthiazoly)‐3‐(4‐sulfophenyl) tetrazolium, inner salt (MTS) and related analogs of 3‐(4,5‐dimethylthiazolyl)‐2,5‐diphenyltetrazolium bromide (MTT) reducing to purple water‐soluble formazans as cell‐viability indicators. Bioorganic & Medicinal Chemistry Letters 1: 611–614.

Berridge MV, Herst PM and Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review 11: 127–152.

Berridge MV and Tan AS (1993) Characterization of the cellular reduction of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Archives of Biochemistry and Biophysics 303(2): 474–482.

Cory A, Owen T, Barltrop J and Cory JG (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Communications 3(7): 207–212.

Gonzalez RJ and Tarloff JB (2001) Evaluation of hepatic subcellular fractions for Alamar Blue and MTT reductase activity. Toxicology in Vitro 15: 257–259.

Hawker JR Jr (2003) Chemiluminescence‐based BrdU ELISA to measure DNA synthesis. Journal of Immunological Methods 274: 77–82.

Henriksson E, Kjellén E, Wahlberg P, Wennerberg J and Kjellström JH (2006) Differences in estimates of cisplatin‐induced cell kill in vitro between colorimetric and cell count/colony assays. In Vitro Cellular & Developmental Biology – Animal 42: 320–323

Marshall NJ, Goodwin CJ and Holt SJ (1995) A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Regulation 5(2): 69–84.

Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65: 55–63.

Muir D, Varon S and Manthorpe M (1990) An enzyme‐linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. Analytical Biochemistry 185(2): 377–382.

Niks M and Otto M (1990) Towards an optimized MTT assay. Journal of Immunological Methods 130(1): 149–151.

O'Brien J, Wilson I, Orton T and Pognan F (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry 267: 5421–5426.

Plumb JA, Milroy R and Kaye SB (1989) Effects of the pH dependence of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐ diphenyl‐tetrazolium bromide‐formazan absorption on chemosensitivity determined by a novel tetrazolium‐based assay. Cancer Research 49(16): 4435–4440.

Raza A, Spiridonidis C, Ucar K et al. (1985) Douglbe laeling of S‐phase murine cells with bromodeoxyuridine and a second DNA specific probe. Cancer Research 45(5): 2283–2287.

Scudiero DA, Shoemaker RH, Paull KD et al. (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48(17): 4827–4833.

Shum D, Radu C, Kim E et al. (2008) A high density assay format for the detection of novel cytotoxic agents in large chemical libraries. Journal of Enzyme Inhibition and Medicinal Chemistry 23(6): 931–945.

Tada H, Shiho O and Kuroshima K (1986) An improved colorimetric assay for interleukin 2. Journal of Immunological Methods 93: 157–165.

Tominaga H, Ishiyama M and Ohseto F (1999) A water‐soluble tetrazolium salt useful for colorimetric cell viability assay. Analytical Communications 36: 47–50.

Wesierska‐Gadek J, Gueorguieva M, Ranftler C and Zerza‐Schnitzhofer G (2005) A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death. Journal of Cellular Biochemistry 96(1): 1–7.

Zhang HX, Du GH and Zhang JT (2004) Assay of mitochondrial functions by resazurin in vitro. Acta Pharmacologica Sinica 25: 385–389.

Further Reading

Berridge M, Tan A, McCoy K and Wang R (1996) The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemical 4: 14–19.

Fan F and Wood KV (2007) Bioluminescent assays for high‐throughput screening. ASSAY and Drug Development Technologies 5(1): 127–136. doi: 10.1089/adt.2006.053.

Mitchison JM (2003) Growth during the cell cycle. International Review of Cytology 226: 165–258. doi: 10.1016/S0074‐7696(03)01004‐0.

Niles AL, Moravec RA, Eric Hesselberth P et al. (2007) A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Analytical Biochemistry 366(2): 197–206. doi: 10.1016/j.ab.2007.04.007.

Riss TL and Moravec RA (2004) Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell‐based cytotoxicity assays. ASSAY and Drug Development Technologies 2(1): 51–62.

Related Sub-Topics:

Cell Biology of Cancer | Cell Death and Cellular Senescence | Techniques and Tools in Molecular Biology | Techniques in Cell Biology | Growth and Synthesis
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Cell Proliferation Assays
 
How to Cite close
Yadav, Kamalendra, Singhal, Nitin, Rishi, Vikas, and Yadav, Hariom(Aug 2014) Cell Proliferation Assays. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002566]