Multi-pass flow cytometry using laser particle barcodes for easy high-marker analysis

Current flow cytometry requires samples to be fully processed and stained for all markers of interest before acquisition. However, assay development become increasingly difficult when many colors (>10) are used at once due to spectral spillover. Assay complexity is hampered further by markers that require special cell processing and restrict the choice of fluorophores or antibodies. We have developed a method to dramatically simplify high-marker flow assays by breaking down a complex measurement into multiple, easier measurements. Our method involves optically barcoding individual cells with semiconductor-based laser particles (LPs) that emit laser light in the infrared. Using LP barcodes and a custom flow cytometer, we track millions of cells across repeated measurements or passes. Cells are de-stained and re-stained after each pass. We demonstrate several applications of multi-pass flow cytometry. First, we show deep immunophenotyping of human PBMCs, measuring 33 markers with 13 colors over 3 cycles. We validate our method against state-of-the-art, reproducing expected cell types and showing improved data quality due to significantly reduced spillover. Second, we show deep characterization of stimulated mouse T cells, in which an unprecedented 32 markers including activation markers, intranuclear proteins, and intracellular cytokines are detected on the same cell type. Finally, we measure cells before and after harsh cell processing steps to recover signals that would otherwise be lost, such as concurrent detection of cytoplasmic GFP and cell cycle. Multi-pass flow cytometry reduces the barrier to high-marker analysis by simplifying complex panels and enabling detection of sensitive signals without compromise. Supported by NIH grant R44GM139504