OMIP‐095: 40‐Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues

Abstract

High‐dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40‐color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co‐stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre‐clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40‐color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.

Cytometry in the Short-Wave Infrared

“…Our SWIR image cytometer can further extend the detection wavelength up to 1550 nm, providing enormous possibilities for biomarker identifications. For example, SWCNTs alone have more than 35 different species that emit at wavelengths in the range of 900–1550 nm, and all of them can be well distinguished using seven different colors of excitations. , An interesting use is to stain and analyze more than 40 biomarkers simultaneously, combined with the conventional visible/NIR cytometer that already allows up to 40 detection channels . Further development of an appropriate detector with an extended spectral window up to 1550 nm for the flow cytometer is needed in the future to achieve the same capability as the SWIR image cytometer.…”

“…The lack of a SWIR-based cytometric tool hinders related research, as current visible-range cytometry cannot be directly applied. Additionally, the most advanced visible cytometry using whole spectral imaging with sophisticated spectral overlap (spillover) compensations has reached its maximum number of detection channels, typically up to ∼20–50 colors. − However, there is a growing need for the simultaneous staining of even more colors, and the unmixing of spectral overlaps becomes extremely challenging, especially with highly overlapped emissions. Expansion of the detection window toward longer wavelengths, , as offered by SWIR, presents a vivid solution.…”

Immune-profiling of T helper 1 (Th1), Th2 and Th17 signatures in murine splenocytes by targeting intracellular cytokines

“…We used CD45 as a common leukocyte marker and gated out dead cells using viability staining (Figure 1 and Online Figure 8). We phenotyped the B cell compartment and subdivided it according to CD19 and CD45R (B220) expression (7). B-1 cells (CD19 + CD45R -) and B-2 cells (CD19 + CD45R + ) are two distinct types of B cells with different functions in the immune system.…”

“…NK cells can recognize and kill cells that are infected with viruses or are transformed (such as cancer cells) without prior sensitization to specific antigens (10). We defined NK cells as CD45 + CD19 -CD45R -CD3 -NK1.1 + in C57BL/6 mice (Figure 1A) according to OMIP-95 (7). Anti-NK1.1 (PK136) antibody does not work in BALB/c mice strains because of an allelic discrepancy of the (11,12), which we also validated (Online Figure 7C) and used anti-NKp46 antibody (10,12) to target NK cells in BALB/c mice (Online Figures 7D and 8A).…”

“…CD62L and CD44 markers are used to define naïve/memory phenotypes (Figure 1A, B and Online Figure 8A, B) in both CD4 + and CD8 + T cell compartments (7). Next, we focused on functional cytokine responses in the non-naïve/effector compartments of both CD4 + and CD8 + T cells after mitogen stimulation.…”