Determining lymphocyte subsets using spectral flow cytometry

Spectral flow cytometry is an innovation in the field of flow cytometry. It differs from conventional flow cytometry by measuring the entire spectrum of light emitted from the sample and using an algorithm to unmix individual signals, rather than using the simple band-pass filters of conventional flow cytometry. We created a 30-colour spectral flow cytometry panel for the purpose of deep phenotyping human lymphocyte subsets, utilising the Cytek Northern Lights 3-laser spectral cytometer. This machine has three solid-state lasers (Violet 405 nm, Blue 488 nm and Red 640 nm), 38 detection channels (V1-V16, B1-B14, R1-R8), and three scatter channels (FSC, SSC-A, SSC-B). Markers were selected to characterise T and B cell differentiation and allow detection of 62 sub-populations in a single tube. Our panel includes lineage markers (CD45 for leukocytes, CD3 for T cells, CD19 for B cells), major subset markers for T cells (CD4, CD8, and γδTCR) and B cells (CD20, IgM, IgD, CD27), and differentiation markers as well as exclusionary markers (CD14 for monocytes and amine-reactive viability dye for dead cells). This talk with discuss the advantages and challenges of using such a panel for lymphocyte phenotyping.