Flow cytometry is the most frequently used high-throughput single cell analysis technology that enables characterization of heterogeneous samples such as blood and bone marrow. It can be used to perform deep phenotyping of immune cells to assess immunological changes in cancer patients or in response to immunizations. The sensitivity and resolution that is necessary to monitor the immune system calls for several optimized multicolor immunophenotyping panels (OMIP). Besides considerable expense and time, this also requires multiple tubes of samples and sensitivity is decreased because the precious blood or bone marrow samples are split between assays. Spectral flow cytometry can analyze a larger number of protein markers because it unmixes the complete emission spectra of individual fluorophores in the panel. Spectral flow cytometry is thus capable of differentiating fluorophores with significant overlap in the emission spectra and enables the use of spectrally similar fluorophores which increases the number of protein markers that can be analyzed.
In a publication in Cytometry, Sahir et al. incorporate 3 CF® Dyes into a spectral flow immunophenotyping panel of 42 surface markers plus viability stain to comprehensively characterize human peripheral blood mononuclear cells (PBMCs). This panel characterizes more than 130 distinct cell populations present in human peripheral blood, including all major immune cell subsets. The authors used Mix-n-Stain™ CF® Dye Antibody Labeling Kits to label several primary antibodies. The use of laminar flow wash technology enabled the authors to stain as few as 2.5 x 105 cells and obtain useful characterization of the PBMCs. This paper develops a feasible workflow for creating an OMIP for extremely high-dimensional flow cytometry. CF® dyes are excellent candidates for spectral flow panels because of their spectrally distinct emission spectra and high quantum yield.
Learn more about Biotium’s reagents for spectral flow cytometry including a growing collection of over 2000 monoclonal CF® dye primary and secondary antibody conjugates, and our Mix-n-Stain™ CF® Dye Antibody and Nanobody Labeling Kits for fast and efficient conjugation to any of our CF® dyes. For cell viability, please view our bright and extremely stable Live-or-Dye™ Fixable Viability Stains available in 14 different colors. Live-or-Dye™ stains 510/550, 665/685, 375/600, and 615/740 are spectrally unique dyes designed specifically for spectral flow.
Full Citation
Sahir F, Mateo JM, Steinhoff M, Siveen KS. Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry. Cytometry. 2020;1–7. https://doi.org/10.1002/cyto.a.24288