Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the <scp>CYTO</scp> 2025 Conference Workshops

Organized by the International Society for Advancement of Cytometry (ISAC), the CYTO conference is one of the most important events for everyone interested in cytometry and quantitative single-cell analysis. It is an annual gathering of cytometry experts and novices in the field, all with a passion for cytometry. This meeting is an opportunity to learn exciting, cutting-edge research directly from the source by attending the Frontiers and State-of-the-Art lectures, research and technology sessions, and Scientific Tutorials. CYTO conferences are also the premier live forum for discussing current and emerging challenges in cytometry and associated sciences through an interactive workshop format. Workshops attract considerable attention within and outside the cytometry community as they address where we stand in specific fields of interest to cytometrists, how current issues with the evolving technologies within the field can be addressed and solved, and what future areas for development exist. Due to the limited time frame allocated to this format, however, workshops run in parallel, preventing attendees from participating in all sessions of interest. Moreover, those unable to attend the CYTO conference miss the content entirely. Therefore, we continue the tradition of summarizing all workshops to disseminate the information provided and to capture the state-of-the-art view of the community. The primary purpose of creating such a combined summary is to preserve the record of live discussions and main conclusions, propose potential guidelines, and make them available to the broader public. The publication of workshop summaries and their availability to a global audience not only captures live discussions but also undoubtedly seeds new initiatives to support emerging topics or trends. This “Cyt-Geist,” a play on the German Zeitgeist, meaning “spirit of the age”, aims to document the defining spirit and collective consciousness of the cytometry field at this moment in time. Just as Zeitgeist captures the prevailing ideas and beliefs of an era, Cyt-Geist captures the current challenges, innovations, and forward momentum that characterize our community. The joint effort begun in 2018 and continued in 2019 by Kamila Czechowska proved to be a valuable reference [1, 2]. Building on that spirit, we present summaries from CYTO 2025, held in Denver, Colorado, from May 31 to June 4. This manuscript serves as a summary report of 15 workshops held at CYTO 2025. We present, in concise form, the current and future challenges in cytometry identified by workshop organizers and participants. The manuscript is organized into three thematic sections: Building the Cytometry Infrastructure of the Future (Standardization, Sharing, and Sustainability in Cytometric Practice); Applied Innovation Across Modalities (Expanding Possibilities: From Fluorescence to Imaging and Automated Annotation); and the People Behind the Panels (Workflows, Workspaces, and the Human Side of Cytometry). Each section addresses critical aspects of modern cytometry practice, from foundational infrastructure and technical innovation to professional development and operational sustainability. We intend to serve with this joint workshop report the global community involved in single-cell analysis and cytometry. Workshops 2, 3, 7, 8, and 13 addressed the foundational frameworks needed to ensure success for the cytometry community in the years ahead. The themes ranged from pre-analytical considerations in sample handling to standardization of instruments and file formats, and from data stewardship to the future of public repositories. Together, these discussions underscored that sustainable progress in cytometry depends not only on innovation but also on building robust infrastructure, shared standards, and reliable practices. WS02 (Pre-analytical variables) examined the numerous factors that can affect the quality of peripheral blood mononuclear cells (PBMCs) and the interpretation of downstream assays, emphasizing the importance of defining the context of use (COU) and minimizing variability. WS08 (Spectral Standardization) highlighted the need for community-driven best practices to account for differences in platforms, reagents, and data analysis methods, including nomenclature and unmixing. WS13 (FCS 4.0) provided historical context for FCS file formats and outlined the community's priorities and timeline for modernizing the standard to support spectral data, interoperability, and high-dimensional analysis. WS03 (FlowRepository) focused on the sustainability and governance of this community resource, emphasizing the need for accessibility, clear licensing, and forward-looking technical development. WS07 (Data Management and Sharing) brought the perspective of Shared Resource Laboratories (SRLs), advocating for broad adoption of the FAIR data principles to support reproducibility, accessibility, and long-term scientific value. Taken together, the outcomes of these workshops point to a central conclusion: the future of cytometry depends on shared responsibility for infrastructure. By harmonizing practices, investing in sustainable platforms, and fostering a culture of open data and reproducibility, the community can ensure that cytometry remains a cornerstone of biomedical discovery. The establishment of task forces, working groups, and continued discussions through community platforms demonstrates the commitment to translating workshop insights into actionable progress. Nicolas Loof, Meredith Weglarz, Jessica Back, Benjamin Daniel, Cheryl Kim, and William Murphy Data management encompasses all disciplines related to handling data as a valuable resource, including annotation, storage, retrieval, security, and organization. A 2014 study [3] demonstrated that the odds of obtaining underlying research data dropped 17% annually after the second year of publication, emphasizing the critical need for effective data stewardship. In cytometry, rapid growth in file sizes from spectral and imaging technologies makes this challenge particularly urgent. 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