Automated antibody dispensing to improve high‐parameter flow cytometry throughput and analysis

Abstract Over the past decade, the flow cytometry field has witnessed significant advancements in the number of fluorochromes that can be detected. This enables researchers to analyze more than 40 markers simultaneously on thousands of cells per second. However, with this increased complexity and multiplicity of markers, the manual dispensing of antibodies for flow cytometry experiments has become laborious, time‐consuming, and prone to errors. An automated antibody dispensing system could provide a potential solution by enhancing the efficiency, and by improving data quality by faithfully dispensing the fluorochrome‐conjugated antibodies and by enabling the easy addition of extra controls. In this study, a comprehensive comparison of different liquid handlers for dispensing fluorochrome‐labeled antibodies was conducted for the preparation of flow cytometry stainings. The evaluation focused on key criteria including dispensing time, dead volume, and reliability of dispensing. After benchmarking, the I.DOT, a non‐contact liquid handler, was selected and optimized in more detail. In the end, the I.DOT was able to prepare a 25‐marker panel in 20 min, including the full stain, all FMOs and all single stain controls for cells and beads. Having all these controls improved the validation of the panel, visualization, and analysis of the data. Thus, automated antibody dispensing by dispensers such as the I.DOT reduces time and errors, enhances data quality, and can be easily integrated in an automated workflow to prepare samples for flow cytometry.