Hybrid Cytometry: automated live-cell tracking with a final snapshot of fixed-cell phenotyping to study immune-synapse dynamics 2263890

Abstract Introduction Even advanced flow cytometry and fluorescence microscopy fall short in linking dynamic immune cell functions and phenotypes at high plexity. We aimed to develop an automated workflow that bridges this gap, capturing mixed culture dynamics with deep phenotyping and optional integration with high-resolution imaging or single-cell RNA sequencing, tracking individual cells throughout the experiment. Methods We implemented an imaging-based workflow integrating automated liquid handling, fluorescence whole-plate scanning, and tracking of sequential activation cycles in live cells, together with corresponding cytokine or surface marker expression in fixed cells. Datasets were computationally aligned to correlate up to 3-plex dynamic functional readouts with phenotypic features. A plexity of 12—20 markers can be achieved through sequential immunostaining. Results This centrifugation-free pericellular liquid exchange workflow enables us to: • Track individual T cells through repeated peptide stimulations of human PBMCs; • Validate activation by downstream cytokine or surface marker staining; • Increase per sample data yield up to 20—100-fold versus ELISpot or flow cytometry; • Visualize transient immune synapses and interactions including antigen presentation, cross-presentation, T-cell—B-cell cooperation, dynamic killing, and Teff—Treg proximity. Conclusion This method provides a scalable, high-content approach for low-volume, single-cell immunology supporting target discovery, vaccine design, and mechanistic research. Funding Source 100XBIO Inc. (Private Investment Funding) Topic Categories Technological Innovations in Immunology (TECH)