Abstract
The dye-dilution assay is a powerful tool to study lymphocyte expansion dynamics. By combining time course dye-dilution experiments with computational analysis, quantitative information about cell biological parameters, such as percentage of cells dividing, time of division, and time of death, can be produced. Here, we describe the method to generate quantitative cell biological insights from dye-dilution experiments. We describe experimental methods for generating dye-dilution data with murine lymphocytes and then describe the computational data analysis workflow using a recently developed software package called FlowMax. The aim is to interpret the dye-dilution data quantitatively and objectively, such that cell biological parameters can be reported with an appropriate measure of confidence, which in turn depends on the quality and quantity of available data.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
de Fries R, Mitsuhashi M (1995) Quantification of mitogen induced human lymphocyte proliferation: comparison of alamarBlue assay to 3H-thymidine incorporation assay. J Clin Lab Anal 9:89–95
Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89:271–277
Hawkins ED, Hommel M, Turner ML, Battye FL, Markham JF et al (2007) Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat Protoc 2:2057–2067
Quah BJ, Warren HS, Parish CR (2007) Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc 2:2049–2056
Hasbold J, Gett AV, Rush JS, Deenick E, Avery D et al (1999) Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester. Immunol Cell Biol 77:516–522
Shokhirev MN, Hoffmann A (2013) FlowMax: a computational tool for maximum likelihood Deconvolution of CFSE time courses. PLoS One 8:e67620
Teodorovic LS, Riccardi C, Torres RM, Pelanda R (2012) Murine B cell development and antibody responses to model antigens are not impaired in the absence of the TNF receptor GITR. PLoS One 7:e31632
Klein AB, Witonsky SG, Ahmed SA, Holladay SD, Gogal RM Jr et al (2006) Impact of different cell isolation techniques on lymphocyte viability and function. J Immunoassay Immunochem 27:61–76
Lyons AB, Hasbold J, Hodgkin PD (2001) Flow cytometric analysis of cell division history using dilution of carboxyfluorescein diacetate succinimidyl ester, a stably integrated fluorescent probe. Methods Cell Biol 63:375–398
Acknowledgment
This work was supported by NIH grant R01 AI132731 (A.H.) and from NIH-NCI CCSG: P30 014195, and the Helmsley Trust (M.N.S.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Roy, K., Shokhirev, M.N., Mitchell, S., Hoffmann, A. (2018). Deriving Quantitative Cell Biological Information from Dye-Dilution Lymphocyte Proliferation Experiments. In: Liu, C. (eds) B Cell Receptor Signaling. Methods in Molecular Biology, vol 1707. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7474-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7474-0_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7473-3
Online ISBN: 978-1-4939-7474-0
eBook Packages: Springer Protocols