Chairs: Axel R Schulz, Claudia Peitzsch
Mass cytometry (Cytometry by time-of-flight, CyTOF) is one of the most powerful tools for high-dimensional single-cell analysis. Due to the lack of signal overlap and cellular autofluorescenceit enables simultaneous measurement of more than 50 parameters in cell suspensions and for spatial analysis of tissue sections. By conjugating antibodies to stable heavy metal isotopes and detecting them by time-of-flight mass spectrometry, the method offers excellent signal resolution, an extensive and expandable marker space, and a comparatively straightforward and rapid panel setup.
In this session, we will briefly introduce the newly formed Mass Cytometry Working Group, which became part of the DGfZ in January 2026. Our invited speaker Ezgi Senoglu from the CRTD in Dresden will present her work on Epi-CyTOF. She used this technique to study epigenetic regulation of human neocortical development in cortical organoids to gain a deeper understanding of the epigenetic deregulation in neuropathologies. In addition, we look forward to contributions from the mass cytometry community that will be selected for oral presentations.
Investigation of the epigenetic complexity of developing human neocortex by Epi-CyTOF
Ezgi Senoglu
CRTD Dresden
The neocortex is the brain structure attributed to higher cognitive functions in humans. Its development is governed by spatiotemporal gene expression programs regulated by epigenetic mechanisms, such as post-translational modifications of histone proteins. Specific histone modifications act on genomic loci to repress or promote gene expression, thereby contributing to the regulation of proliferation and differentiation of neural progenitor cells. Given the large number of histone modifications, we currently lack an understanding of the epigenetic state of neural cell populations beyond a few well-studied histone modificaitons. Moreover, the interplay of these modifications and their temporal changes in the developing human neocortex remain largely uncharacterized. To unravel this combinatorial crosstalk and complexity of the epigenome during neurogenesis at single-cell resolution, we employ cytometry by time of flight with a comprehensive epigenetic panel spanning over 30 different epigenetic markers, referred to as Epi-CyTOF, and cell type markers covering different neural cell types. Our data proves that Epi-CyTOF can detect and distinguish distinct neural cell populations in human foetal tissue and human cortical organoids. Furthermore, it reveals cell-type specific distribution of histone modifications. Epi-CyTOF presents a powerful technology to decipher the complexity and dynamics of histone modifications during brain development and can in the future be applied to elucidate epigenetic changes in human neurodevelopmental disorders caused by mutations in epigenetic modifiers.
Multisite Harmonization of Mass Cytometry Workflows for Translational Immune Profiling
Mehmet Serdar Koca1, Katrien L.A. Quintelier2,3, Lucía Rodríguez-Doña1, Adrián Barreno4, Axel Schulz4, Sonia Gavasso5,6, Patrice Hemon7, Divi Cornec7, Sofie Van Gassen2,3, Sarah Bonte2,3, Yvan Saeys2,3, Marta E Alarcón-Riquelme1,8, Concepción Marañón*1, Paulina Rybakowska*1
1 Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain
2 Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Ghent, Belgium
3 Data Mining and Modeling for Biomedicine Group, VIB Center for Inflammation Research, Ghent, Belgium
4 Mass Cytometry Lab, German Rheumatism Research Center (DRFZ), A Leibniz Institute, Berlin, Germany
5 Department of Clinical Medicine, University of Bergen, Bergen, Norway
6 Department of Neurology, NeuroSys-Med, Haukeland University Hospital, Bergen, Norway
7 B Lymphocytes, Autoimmunity, and Immunotherapies, UMR1227, Immunology Department, Augustin Morvan Hospital, Brest, France
8 Institute for Environmental Medicine, Karolinska Institute, Stockholm, Sweden
*Equal contributions
Keywords: CyTOF, Multicenter study, Harmonizations, Immune monitoring
Background: Harmonized workflows are essential for reproducible mass cytometry (CyTOF) data in multicenter clinical trials. This study evaluates optimization strategies for sample preservation, acquisition conditions, and compensation to ensure inter-center comparability.
Methods: Three barcoded samples were stained with 25- or 46-plex antibody panels. Signal stability during long acquisition was assessed across different acquisition solutions (CAS, CASPlus, Water) and post-thaw storage conditions (4°C/−80°C for up to 1 month). Compensation robustness was tested using single-stained compensation beads stored frozen for up to 1 year or subjected to freeze-thaw cycles. Stained samples and compensation beads were distributed across four acquisition centers over three batches to evaluate inter-center reproducibility. Cell frequencies (CF) and median signal intensity (MSI) values were analyzed.
Results: CASPlus provided the most stable signal intensities during long acquisition compared with CAS and Water. CSB and CASPlus at 4°C caused solution-dependent cell depletion and marker redistribution. In contrast, stained samples frozen at −80°C remained stable for up to 2 months, maintaining low coefficients of variation (<15% for CF and <20% for MSI). Compensation beads remained highly stable for up to 1 year, and a centralized compensation matrix successfully corrected spillover across all sites. Inter-center analyses of CF and MSI consistently distinguished donor samples, while intra-sample variability was further reduced by separate analysis of PBMC and granulocyte.
Conclusions: Optimization of acquisition solutions, sample and compensation bead preservation, and centralized compensation matrix calculation enables robust harmonization of CYTOF data across centers, supporting reliable immune monitoring in multi-center clinical studies.
IMI: PRECISESADS(GA#115565), 3TR(GA#831434), SIGNATURE(GA#101072891)
SceniTOF - Functional single-cell multiplexed metabolic profiling to map bioenergetics of heterogeneous immune cells by mass cytometry in mice and men
Niclas Schierloh1*/Laura Riechert2,3*/Anna-Lena Schäfer2,3*, Emilia Schlaak1, Ke Meng1, Reinhard E. Voll2,3, Nina Chevalier2,3*/Bertram Bengsch1,4*
1Department of Internal Medicine II, Medical Center – University of Freiburg, Freiburg, Germany
2Department of Rheumatology and Clinical Immunology, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
3Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
4Signalling Research Centres BIOSS and CIBSS, Freiburg, Germany
* equal contribution
Immune cell activation, differentiation, and effector function are tightly coupled to cellular metabolism, but many functional metabolic assays lack single-cell resolution. Single-cell metabolic regulome profiling (scMEP) by mass cytometry quantifies metabolic proteins alongside immunophenotypes, but protein levels do not necessarily reflect metabolic activity or flux. The method SCENITH (Single Cell ENergetIc metabolism by profilIng Translation inHibition), developed by flow cytometry, may overcome this limitation by quantifying pathway-specific effects on protein synthesis after brief ex vivo inhibitor treatment.
Here we established a mass cytometry-compatible version of the SCENITH assay using a secondary metal-coupled antibody to detect the translation inhibitor puromycin as an integral part of the SCENITH assay and combine it with the scMEP approach. Briefly, human or murine immune cells are treated ex vivo or after in vitro stimulation with inhibitors of glycolysis and oxidative phosphorylation, pulsed with puromycin, barcoded, and stained with a multiplexed CyTOF panel targeting metabolic regulators, lineage markers, and activation states. This combined SCENITH–CyTOF approach enables scalable mapping of energetic pathway dependence while concurrently resolving phenotypic and metabolic profiles. We demonstrate the applicability of the SceniTOF assay to identify the differences in metabolic state and flux across human T cell differentiation states and the murine B cell compartment.
Together, this integrated approach links multiplexed single-cell phenotyping with functional metabolic readouts, allowing SceniTOF to relate metabolic potential to actual metabolic flux by mass cytometry.