OMIP-050: a 28-color/30-parameter fluorescence flow cytometry panel to enumerate and characterize cells expressing a wide array of immune checkpoint molecules.
OMIP-044: 28-color immunophenotyping of the human dendritic cell compartment. High-dimensional single cell analysis identifies stem-like cytotoxic CD8(+) T cells infiltrating human tumors.
Background fluorescence and spreading error are major contributors of variability in high-dimensional flow cytometry data visualization by t-distributed stochastic neighboring embedding. Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells. Data analysis in flow cytometry: the future just started. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis.īendall, S. We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system.
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