Multiplication and Division: Cell Proliferation Monitoring by Dye Dilution Analysis

The Speakers

Katharine A. (Kathy) Muirhead, PhD - Chief Operating Officer, SciGro Inc.

Kathy Muirhead, PhD is Chief Operating Officer of SciGro, Inc., which she co-founded in 1996. In prior lives she served as Assistant Professor of Pathology at the University of Rochester, director of the first flow cytometry core facility at SmithKline Beckman R&D, and Senior V.P. of Research & Business Development at Zynaxis, Inc., a cell therapy start-up co-founded with colleagues from SmithKline. Dr. Muirhead has served as an ISAC Councilor, Associate Editor and reviewer for Cytometry A, and on CLSI subcommittees formulating guidelines for clinical immunophenotyping and validation of flow cytometric assays. While at the University of Rochester she co-founded the Annual Courses in Applications of Cytometry, a “for the community, by the community” educational series now nearing its 50^th anniversary. She currently serves on the ISAC Governance Committee and the Board of Directors of Cytometry Educational Associates, Inc.  

Paul K. Wallace, PhD - CSO, SciGro, Inc, Southwest Office, Roswell Comprehensive Cancer Center Professor Emeritus

Paul K. Wallace served from 2003 to 2021 as Director of the Flow and Image Cytometry Department at Roswell Park Comprehensive Cancer Center (RPCCC) in Buffalo, NY, where he is now Professor Emeritus. He is the current Educator-in-Chief and a Past President of the International Society for Advancement of Cytometry, an organization. Dr. Wallace is also Chief Scientific Officer of SciGro, Inc., a biomedical consulting group. He remains active in the International Clinical Cytometry Society, serves as Associate Editor of Clinical Cytometry B, and in 2018 received their Wallace H. Coulter Award for lifetime achievement in clinical cytometry.

Summary
Cytometrists have long been interested in the biology of proliferation, both in normal growth and in tumors. Flow cytometric methods to measure proliferation fall into two main categories: those that assess DNA content or cell cycle status, and those that track cell division by dye dilution.

Cell cycle analysis is a well-established application. S-phase fraction, for example, indicates the proportion of cells actively synthesizing DNA at a given time. Combining DNA binding dyes with phase-specific markers allows discrimination between cells with identical DNA content that are actively cycling (G1) or quiescent (G0). However, these methods don’t reveal how many times a cell has divided in response to stimulation, how much a particular subset has expanded, or what fraction of the starting population has participated in the response.

In contrast, dye dilution allows more direct monitoring of cell division. Cells are labeled with bright, stable, non-toxic fluorescent dyes that bind to proteins or membranes and are distributed approximately evenly between daughter cells at each division. The resulting fluorescence intensity profile reflects the proportion of responder cells in the starting population and the number of cell divisions undergone by each responder during the assay period. This approach has been widely applied to in vitro proliferation studies, antigen-specific cell enumeration, and the identification of quiescent stem cell populations.

After a brief overview of DNA-based methods, this presentation will focus on dye dilution methods for proliferation monitoring, highlighting dye-specific considerations, analysis strategies, critical controls, technical challenges, and key applications. 

Learning Objectives:
After attending this webinar, participants will be able to:
1.  Compare approaches for monitoring proliferation by DNA cell cycle analysis and dye dilution, including the strengths and limitations of each.
2.  Select appropriate dyes and establish the necessary experimental controls and analysis strategies for reliable dye dilution assays.
3.  Describe how dye dilution can be used to quantify low frequency populations of interest, including antigen-specific T cells or quiescent/stem-like tumor cells.

Who Should Attend:
SRL Staff, Biologists interested in measuring proliferation, Cancer biologists, Immunologists, Individuals doing infectious disease & vaccine research, Individuals involved in R&D and manufacturing of cellular therapeutics 

CMLE Credit: 1.0