Flow Cytometry Webinars

Applications of imaging flow cytometry for the diagnosis of leukaemia


Wendy Erber, Consultant Haematologist and Professor of Pathology and Laboratory Medicine, The University of Western Australia; and PathWest Laboratory Medicine

Kathy Fuller, UWA Senior Research Fellow and PathWest Medical Scientist in Charge, Translational Cancer Pathology Laboratory, The University of Western Australia; and PathWest Laboratory Medicine


The diagnosis, classification and prognostic stratification of leukaemia requires incorporation of morphology, immunophenotype and genetic data. These are performed on a number of platforms, often in separate laboratories, and the results integrated.

Imaging flow cytometry has opened new opportunities in the assessment of leukaemia whereby this information can be generated on one automated instrument: an imaging flow cytometer. Image-based flow cytometry combines high-resolution digital images with the quantitative fluorescence information gained from a standard flow cytometer. The imaging functionality allows the localisation of cellular antigens to be determined (i.e. cell surface, cytoplasm, nuclear), a feature that cannot be achieved by standard flow cytometry. Specific cell populations can be selected for analysis based on combined variables such as fluorescent intensity, cell shape, cell size and texture.

In this webcast the value of imaging flow cytometry for the assessment of acute and chronic leukaemias will be demonstrated. We will look at the detection of disrupted PML protein in acute promyelocytic leukaemia and the ability to distinguish between cytoplasmic and nuclear NPM in acute myeloid leukaemia. The webinar will also present exciting new data demonstrating the ability to detect chromosomal abnormalities by FISH in phenotyped leukaemia cells in chronic lymphocytic Leukaemia.

During this webcast you will learn about the applications of imaging flow cytometry for the assessment of acute and chronic leukaemia. This will be illustrated by being able to visualize:

  • Abnormal patterns of protein expression in leukaemia cells
  • Cellular localisation (cytoplasmic versus intranuclear) of proteins in leukaemia cells
  • Detection of chromosomes by FISH in leukaemia cells identified by their phenotype
  • Methodology to perform imaging flow cytometry in a clinical laboratory

ToxTracker Assay for High-throughput Screening of Genetic Toxicity

Presenter: Giel Hendriks, CEO, Toxys


With the increasing production of new chemicals for a wide range of applications in health care, food and cosmetics comes the demand for rapid and reliable approaches for toxicity assessment. Novel innovative in vitro systems should ideally not only identify toxic properties of chemicals, but also provide insight into the type of cellular damage inflicted in order to more reliably predict human health hazard of novel compounds.

ToxTracker is a mammalian stem cell-based reporter assay that detects activation of specific cellular signalling pathways upon exposure to unknown compounds (Hendriks et al, Tox Sci 2016). ToxTracker contains six different GFP-tagged reporters that allows discrimination between induction of different types of DNA damage, various cellular anti-oxidant responses and activation of the unfolded protein response. The ToxTracker reporter cell lines are combined in 96-well plates, exposed to the test samples, typically for 24h, and induction of the fluorescent reporters is examined by flow cytometry (Guava® easyCyte).

The integrative approach of the ToxTracker assay provides a powerful tool for in vitro cancer hazard screening of chemicals by unveiling activation of specific cellular signalling pathways upon exposure and deliver insight into the underlying mechanism of toxicity.

In this webcast you will learn:

  • A novel approach to toxicity screening
  • How the unique microcapillary fluidics design of the Guava® flow cytometers enables direct sampling, conferring the distinct advantage of absolute cell counts, and permitting direct correlation between cellular responses and cytotoxicity

Monitoring Immune Function by Imaging Flow Cytometry

Presenter: Orla Maguire, Ph.D., Roswell Park Cancer Institute, Buffalo, NY


The immune system plays a critical role not only in fending off pathogen attack, but also in cancer surveillance, and more recently as a tool in immunotherapy-based treatments. Immune cell functions are tightly regulated by essential transcription factors such as NF-κB and NFAT. Monitoring immune cell activity – including phenotyping immune cell subsets, tracking cell proliferation, and measuring cytokine production – can provide insights into the overall status of immune function in patients, particularly those undergoing immunosuppression after transplants, enduring cancer treatment, or suffering from autoimmune disease or other pathologies that affect the immune system. Imaging flow cytometry (IFC) has emerged as a useful and efficient tool for studying the signaling pathways in immunophenotypically defined subpopulations of immune cells. This technique enables quantitative image analysis of the intracellular localization of the signaling intermediaries NF-κB and NFAT as parameters of immune activity. This webinar will introduce viewers to the process of using IFC to determine subcellular localization of biomarkers, including a discussion of how IFC can help to assess the activity of transcription factors, or the drug-induced stimulation or inhibition thereof, in clinical samples.

What will you learn? During the webinar, viewers will:

  • Learn the process of using IFC to study transcription-factor signaling
  • Discover how IFC can help to determine the effects of drug inhibition or stimulation on immune function
  • Gain insight into preclinical and clinical applications of IFC
  • Be able to ask questions during the live broadcast!

Circulating Tumour Cells: Liquid Biopsy by High-throughput, High-resolution Imaging Flow Cytometry

Presenter: Dr. David Jamieson, Research Associate Northern Institute for Cancer Research, University of Newcastle upon Tyne


Circulating tumour cells (CTCs) have the potential to act as a source of tumour tissue for the measurement of pharmacodynamic biomarkers in early phase clinical trials. This webcast will describe the sample processing, data collection and data analysis used by Dr. David Jamieson to characterise CTCs by imaging flow cytometry.

Live Cell Imaging Reveals How Cells Find and Maintain Their Division Sites

Presenter: PJ Buske, PhD Postdoctoral Research Fellow Lab of Dr. Dyche Mullins, HHMI/UCSF


Knowing when and where to divide is essential to cell survival and to guarantee proper allotment of the cellular contents in order to produce viable daughter cells. In bacteria, the division site is marked by the formation of a cytoskeletal ring (Z ring) made up of filaments comprised of the highly conserved tubulin homolog FtsZ. Once assembled, the Z ring serves as a platform for assembly of the division machinery. To date, how anchoring of FtsZ filaments to the membrane occurs has been poorly understood.

In this webinar, Dr. PJ Buske will describe how dual-color, continuous live-cell fluorescent imaging using the CellASIC® Onix2 system has allowed the Mullins lab at UCSF to track co-localization of FtsZ and anchor proteins over multiple division cycles. Dr. Buske will discuss how this technology can be used to elucidate key protein interactions with FtsZ to drive filament dynamics and other processes such as cell wall synthesis and DNA replication.

Autophagy in the Hematopoietic System

Presenter: Dr. Anna Katharina (Katja) A Simon, Kennedy Institute of Rheumatology, University of Oxford


Autophagy is essential in determining cell fate in hematopoietic cells. Firstly the removal of mitochondria from erythroblasts allows survival and their final maturation step into mature red blood cells. Secondly autophagy is essential to strike the fine balance between quiescence, self-renewal, and differentiation of hematopoietic stem cells. Thirdly declining levels of autophagy in ageing macrophages contributes to senescent features. And lastly we have shown that memory T cells, the cells that mediate long-term protection after vaccination, cannot form in the absence of autophagy as they fail to undergo a necessary metabolic switch. We have also established different ways to detect autophagy in primary immune cells and rare stem cells using the ImageStream® imaging flow cytometer and traditional flow cytometry. With these techniques we have been able to contribute to the description of the first immune deficiencies in which the autophagy/lysoosomal pathway seems to be involved.

In the long term, understanding molecular mechanisms that can manipulate cell fate of hematological stem cells and their progeny will be essential in the context of hematological malignancies, regenerative medicine and immune responses.

In this webinar you will learn:

  • How autophagy influences hematopoietic cell survival and cell fate.
  • Macrophage senescence is associated with decreased autophagy.
  • The best ways to measure autophagy in primary cells.

Analysis of Extracellular Vesicles Including Exosomes by Imaging Flow Cytometry

Presenter: André Görgens, Ph.D. University Hospital-Essen, Essen, Germany


Extracellular vesicles (EVs) such as exosomes (70 nm – 160 nm in diameter) and microvesicles (100 nm – 1,000 nm diameter) can be harvested from cell-culture supernatants and from all bodily fluids. Current standard techniques to visualize, quantify, and characterize EVs are electron microscopy, nanoparticle tracking analyses, and dynamic light scattering. To further characterize and discriminate EVs, more exact high-throughput technologies to analyze their surface are highly desired. Although conventional flow cytometry is limited to measuring particles down to approximately 300 nm – 500 nm, a relatively new flow-cytometric method—called “imaging flow cytometry”—allows for the analysis of EVs smaller than 300 nm. This webinar will introduce viewers to the challenges, limitations, and pitfalls of flow cytometry-based EV analysis, and to the imaging flow cytometry methodology. Also covered will be techniques for analyzing exosomes, microvesicles, and apoptotic bodies in unprocessed samples, how imaging flow cytometry can be used to evaluate or reevaluate EV isolation techniques, and the advantages and disadvantages of using this method.

Quantitating Critical Subcellular Events in Significant Populations Using Imaging Flow Cytometry

Presenters: Olivier Thaunat, M.D., Assistant Professor, Hôpital Edouard Herriot, Hospices Civils de Lyon, University of Lyon
Christopher Parris, Ph.D., Director, Division of Biosciences-Life Sciences, Brunel University London, College of Health and Life Sciences


During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Imaging flow cytometry reveals that antigen polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.

Rigorous methods for the detection of DNA-damaged foci in eukaryotic cells are fundamental to DNA repair studies, which delineate mechanisms and may identify differences in DNA repair capacity among cell types. The Parris lab performed the first analyses to demonstrate the use of imaging flow cytometry for the detection of ã-H2AX foci in cells exposed to ionizing radiation that induces DNA double strand breaks. This webinar presents data validating the enhancement of foci quantitation and image resolution employing imaging flow cytometry, using cell lines derived from normal individuals. In this webinar, Dr. Thaunat’s work demonstrates that imaging flow cytometry (IFC) is key to the identification of polarized distribution of antigen on B cells that persists in vivo. Dr. Parris will describe how IFC with multimagnification and extended depth of field enhances quantitation of DNA damage in cells, providing a quantitative alternative to traditional low-throughput in situ microscopy methods for the detection of ã-H2AX foci.