Abstract
This protocol was developed to increase the richness of information available from in vivo T cell proliferation studies. DNA labelling techniques such as BrdU incorporation allow precise control of label administration and withdrawal, so that the division history of a population can be tracked in detail over long timeframes (days-weeks). Ki67 is expressed in the nucleus of dividing cells, and is retained for a short time (3-4 days) after division (Gossel et al., 2017); therefore acting as a molecular clock to identify cells that have recently divided. Combining these two techniques allows the integration of current and historical proliferation information from individual cells within a population. This data can subsequently be used to probe population dynamics by fitting mathematical models of proliferation (Gossel et al., 2017).
Keywords: T cells, Ki67, BrdU labelling, T cell proliferation, DNA labelling, Flow cytometry
Background
Quantifying the dynamics of T cell death and division is a significant experimental and computational challenge, yet this information is vital to our understanding of how a healthy immune system develops and is maintained. A variety of techniques exist for in vivo analysis of T cell population dynamics, but the interpretation of data is complicated and results can be model-dependent (Asquith et al., 2002; De Boer and Perelson, 2013). Many of these assays are based on flow cytometric readouts, providing an opportunity to gain novel insights by exploiting the potential for multiparametric analysis. Combining analysis of BrdU uptake alongside Ki67 expression is particularly useful as each represents a distinct parameter of cell division. BrdU is a thymidine analogue that is incorporated into the DNA of dividing cells, and thus permanently marks cells that have divided during the labelling period (Tough and Sprent, 1994; Bonhoeffer et al., 2000). In contrast, Ki67 is a transient marker of cell division: the Ki67 protein is expressed during cell cycle and for a short duration (3-4 days) thereafter (De Boer and Perelson, 2013; Gossel et al., 2017). Measuring Ki67 expression simultaneously with BrdU uptake therefore provides both recent and historical proliferation information from the same cell.
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This protocol was developed for a published study (Gossel et al., 2017), supported by the National Institutes of Health (R01 AI093870) and the Medical Research Council (MC-PC-13055). The authors declare no conflicts of interest or competing interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.