Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

Dielectrophoresis-assisted creation of cell aggregates under flow conditions using planar electrodes.

We present a microfluidic platform allowing dielectrophoresis-assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom-up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell-cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.