![]() ![]() Highly multiplexed measurements of immune cells to characterize dozens of different cell types have proven to be critical in the development of immunotherapies and vaccines 3– 6, as well as detection of minimal residual disease in leukemia 7. While these are major advantages in throughput and cost 1, 2 over technologies such as single-cell mass cytometry and sequencing-based proteomic analysis, flow cytometry is facing significant challenges in meeting the growing demand to measure more protein markers per cell. Continuing advances in high-speed fluidics and multi-color optics, as well as fluorophore chemistry, has enabled high-parameter measurement (up to ~30 markers) at high speed (>10,000 cells per second) and low cost. Our results open a new avenue in multi-dimensional single-cell analysis based on optical barcoding of individual cells.įluorescence-based flow cytometry has been a workhorse in the single-cell analysis of surface markers, intracellular cytokines, intranuclear proteins such as transcription factors, and cell cycles. This workflow allowed us to use only 10-13 fluorophores in each cycle, significantly reducing spectral spillover and simplifying panel design. Second, we show 33-marker deep immunophenotyping of PBMCs, analyzing the same cells in 3 back-to-back cycles. First, we demonstrate unprecedented time-resolved flow characterization of T cells before and after stimulation. We demonstrate the benefits of this approach on several pertinent assays with human peripheral blood mononuclear cells (PBMCs). Here, we present multi-pass flow cytometry, in which cells are tracked and measured repeatedly through barcoding with infrared laser-emitting microparticles. ![]() However, the overlap of fluorophore emission spectra and one-time measurement nature of flow cytometry are major barriers to meeting the need. Recent advances in immunology and immuno-oncology as well as drug and vaccine discovery have increased the demand to measure more parameters. Flow cytometry is a standard technology in life science and clinical laboratories used to characterize the phenotypes and functional status of cells, especially immune cells.
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