By IU Simon Comprehensive Cancer Center
Dec 06, 2024
Flow Cytometry Q&A
Baohua Zhou, PhD, associate professor of pediatrics at IU School of Medicine, is director of the cancer center’s Flow Cytometry Core. He answers questions about the core and how it benefits cancer center members.
Q: First off, what is flow cytometry?
A: Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through beams of lasers with different wavelength. The properties measured include a cell’s relative size, relative granularity or internal complexity, and relative fluorescence intensity of markers (proteins) expressed by a cell.
Q: What are the most common research questions that can be answered with the flow core's technology?
A: The instruments in the flow core allow cancer center researchers to investigate the development and function of the immune system in health and disease, aspects like hematopoietic malignancies and immune cell populations and phenotypes in tumor microenvironment (immunophenotyping). The technologies in the core can also help researchers to identify and validate new targets for cancer treatment.
The Cytek Aurora has helped me to significantly expand the complexity of my panels, allowing for more comprehensive multiparameter analyses without compromising data quality. Its spectral unmixing capabilities have streamlined my workflow, saving valuable time during data acquisition and analysis. Additionally, the system’s sensitivity and flexibility have enabled me to explore novel markers and uncover deeper insights in my research.
Wei Luo, PhD
Q: How would you describe the core to cancer center researchers?
A: Flow cytometry is integral and critical for the research activities of many of the cancer center members. As a centralized facility, the Flow Cytometry Core provides cancer center researchers advanced resources of state-of-the-art flow cytometric technologies.
Q: The core has several instruments that perform flow cytometric assays. Can you give us an overview of those instruments and what they do?
A: The core has several different types of instruments as described below:
Flow cytometric analyzers: Used to analyze surface marker expression, intracellular protein levels, cell cycle phase, cell viability, apoptosis, proliferation rates, and the identification of specific cell subsets within a sample.
Flow sorters: Used to purify (sort) particular type(s) of cells of interest from a sample containing many different cell types for further analysis or study. The sorted cells of interest can be either collected in a tube (bulk sorting) or deposited individually into each well of 96 well plates. In bulk sorting, four cell populations from a sample can be sorted simultaneously.
Imaging flow cytometer: This instrument is a combination of flow cytometric analyzer and fluorescence microscope. In addition to the analyses of cells described in the flow cytometric analyzers described above, the imaging flow cytometer will also simultaneously take multiple pictures (brightfield and fluorescent) of a cell flow through the fluid stream. Measuring both expression levels and spatial variations of the proteins, this instrument can be used to study cell signaling, protein co-localization, cell-cell interaction, protein aggregation, and much more.
Q: What services does it provide to cancer center researchers?
A: The flow core provides cancer center researchers hands-on training on flow analyzers, staff-assisted cell sorting, and consultations on panel design and trouble shooting.
I’ve used the Cytek Aurora to do the immune profiling analysis in the preclinical models of breast cancer, colorectal cancer, and pancreatic cancer. It also helped us to capture the change of tumor immune microenvironment when we knockdown some key genes in the tumor immune response pathway. That’s very useful technology.
Xinna Zhang, PhD
Q: Let’s focus on another instrument, the Cytek Aurora. What is it and what does it do?
A: Cytek Aurora is a spectral flow cytometer newly acquired by the flow core. While conventional flow cytometric analyzers collect fluorescent emission data in discrete wavelengths, thus cannot discriminate fluorophores with emission peaks close to each other; Aurora integrates innovative technology to collect fluorescent emission data in full spectrum (thus spectral flow cytometry) allowing the individual resolution of fluorophores with similar emission spectra. This technology provides unprecedented flexibility to scientists to use a wide array of new fluorochrome combinations and the number of markers possible in a multicolor panel is greatly expanded. We have successfully designed a panel of 23 markers to phenotype tumor infiltrating T cells.
Q: Are there any success stories or unique cases from researchers who have used flow that you’d like to mention?
A: Flow cytometry is one of the most popular technologies used in studying immune system development and function, infectious diseases, pathophysiology of immune-mediated diseases (allergy and autoimmunity), and recently cancer immunotherapy. There are many success stories from cancer center researchers using the flow core. For example, researchers in the lab of Mark Kaplan, PhD, used cell sorter in the core to purify macrophages from tumor-bearing mouse lungs and transfer these cells to recipient mice to study the role of macrophages in lung cancer development and metastasis. Maegan Capitano, PhD, used the Image Stream in the core to study NF-κB signaling pathway and irradiation-induced DNA damage, apoptotic signaling and early cycling of hematopoietic stem cells.
Questions about the Flow Cytometry Core? Contact Baohua Zhou, PhD, core director, at zhoub@iu.edu.
