Session 5 – Assays, reagents and functional probes: New methods in flow cytometry
Friday, 12.09.2025, 10:45 am – 12:00 pm
Chair: Bastian Höchst
Flow cytometry is a powerful tool for the analysis of cells and particles. This session will focus on the latest developments in assays, reagents and functional probes that enable scientific questions to be answered accurately and efficiently. The aim of this session is to inform participants about innovative methods and technologies that extend and improve the application of flow cytometry. We will discuss specific topics such as developing new assays, optimising reagents, and introducing functional probes to study cellular processes. Emphasis will be placed on the practical application of these methods to address complex scientific questions. This session will be of great interest to researchers, technicians and students involved in the development and application of assays, reagents and functional probes in flow cytometry. Attendees will gain valuable insights into the latest scientific advances and practical applications that can enrich their own work and research.
Philippe Pierre
Head of the Immunology Department at the Centre d’Immunologie de Marseille-Luminy, France
Monitoring protein synthesis as an indicator of cell activation and homeostasis.
Monitoring protein synthesis using puromycilation and flow cytometry allows for the study of cell activation and metabolic responses in multiple cell subsets in parallel. We now have developed SNUPR (Single Nuclei analysis of the Unfolded Protein Response), an accessible technique that allows the profiling of the three UPR branches in nuclear suspensions by flow cytometry, and applied it to study UPR dynamics in a cancer-specific context. We detected in different human cancer cell lines, a high heterogeneity in UPR activation, which could not have been predicted simply by monitoring individual sensors’ expression levels. SNUPR analyses further indicate that this heterogeneity is explained by variations in the intensity and duration of ER stress-induced protein synthesis inhibition via PERK, acting as upstream regulator of both the IRE-1/XBP1 and ATF6 dependent transcriptional programs. We extend the relevance of these observations by demonstrating that IRE-1/XBP1s pathway plays a critical role in bortezomib resistance of multiple myeloma cells and patients. SNUPR can be used to monitor UPR dynamics with single-cell resolution and identified clinical contexts in which targeting a specific UPR branch could be detrimental or help circumventing chemotherapy resistance.
Biosketch
Dr. Philippe Pierre is an international leader in dendritic cell biology and innate immunity research. During his PhD at the EMBL (Heidelberg, Germany), his research focused on the characterization of the microtubule-binding protein CLIP-170. After a post-doctoral research period at Yale University School of Medicine (USA), he focused on MHC II-restricted antigen presentation and immune response. In 2000, he created his laboratory at Centre d’Immunologie de Marseille-Luminy (CIML, France) focused on the cell biology of dendritic cell activation with projects studying proteolysis, membrane trafficking, stress pathways, autophagy and microbial detection, while initiating a systemic analysis using genomic and proteomic technology. The team was first to demonstrate the importance of the ubiquitin ligase MARCH1 and the small RNA miR-155 in controlling DCs activation. The observation that regulation of mRNA translation and induction of stress pathways are central to DC function, motivated its most recent work on the role of the integrated stress pathway during microbial detection by DCs. The laboratory has also developed new technologies to monitor protein synthesis, energy metabolism and the unfolded protein response by flow cytometry that are now used worldwide. Dr. Pierre has published over 120 articles in peer-reviewed journals (including Nature, Cell, Immunity, EMBO J., PNAS, Nature Com, Cell Metabolism). P. Pierre received the “Jean-Marie Legoff 2015 Prize” for molecular immunology from the French Academy of Sciences. He was the director of the CIML until 2024 and is professor-adjunct of the University of Aveiro (IBiMED, Portugal) and the Shanghai Institute of Immunology (SII, PRC).
Eric Sündermann
Eric Sündermann1,2, Bob Fregin1,2, Doreen Biedenweg1, Jan Maurice Wilder1 and Oliver Otto1,2
1Institute of Physics, University of Greifswald, Greifswald, Germany
2DZHK Partner Site Greifswald, Greifswald, Germany
A fast cytometric approach to assess the membrane tension of cells and mitochondria in situ
Current research emphasizes the importance of cell mechanics to understand cell state and function. Recently, the focus shifted from mechanical bulk properties to membrane tension, which plays a key role in various pathophysiological processes, e.g., the host-pathogen interaction in immune response. However, traditional methods that measure membrane tension are not able to provide sufficient throughput to screen entire cell populations or resolve dynamic processes within a cell. Furthermore, direct measurements of membrane tension on a sub-second timescale are widely unknown.
Recently, we introduced membrane tension cytometry (MTC) for analysing the membrane tension of suspended cells on a millisecond timescale, resulting in a throughput of 100 cells per second. MTC integrates flow cytometry and fluorescence lifetime analysis, utilising a membrane tension-sensitive dye (Flipper-TR). We established a novel calibration strategy for Flipper-TR on red blood cells (RBCs) based on real-time deformability cytometry of osmotically stressed RBCs. In addition, we studied human leukaemia cells (HL60), aiming to disentangle membrane and bulk contributions to the mechanical properties of cells. We depleted cholesterol in the plasma membrane and inhibited actin polymerisation. While we observed a reduction in fluorescence lifetime but no change in bulk mechanics when cholesterol was depleted, interference with the cytoskeleton impacts bulk stiffness only.
Finally, we combined MTC with a mitochondrial membrane tension-sensitive dye (Mito Flipper-TR) to directly measure mitochondrial membrane tension inside cells. Our results show that hydrodynamic stress, propagates through the cytosol and increases mitochondrial membrane tension. This high-throughput, in situ monitoring is crucial for understanding cellular pathophysiology, as mitochondrial membrane tension is linked to their fission capability, which plays a critical role in cell function under metabolic or environmental stress.
Thomas Middelmann
Thomas Middelmann, Yannik Hein, Lorenz Mitschang and Martin Hussels
Physikalisch-Technische Bundesanstalt (PTB), Berlin, Germany
Towards determination of the antibody binding capacity from real-time measurements of reaction kinetics in a flow cytometer
The determination of the cellular antibody binding capacity (ABC) by flow cytometry is an accepted technique for the characterization of cells in health and disease. But the quantification of the ABC is typically based on reference measurements with calibration-beads to estimate the detection efficiency of the fluorescence intensity. An alternative approach is the absolute ABC quantification based on the evaluation of the reaction kinetics, as has been presented by Moskalensky et al. 2015 [1] and Khalo et al. 2023 [2]. It can be independent of the fluorescence intensity calibration and may be more robust than the calibration-beads-based approaches. We consider the implementation of this approach in real-time measurements of the reaction kinetics in a state-of-the-art flow cytometer, which would allow us to reduce the number of measurements and preparation steps required. We estimate the parameter space in which this is beneficial and analyze connected challenges and advantages. Based on exemplary measurements with Alexa Fluor™ 488 labeled anti-CD4 antibodies on lymphocytes we discuss extensions of our setup and derive protocols for preparation, measurement and evaluation.
[1] https://doi.org/10.1016/j.jim.2015.02.001
[2] https://doi.org/10.1016/j.jim.2023.113555