Clec12A, CD301b, and FcγRIIB/III define the heterogeneity of murine DC2s and DC3s

Protocol for mapping the heterogeneous dendritic cell network across the murine tissue landscape via high-dimensional flow cytometry

“…Within all tissues, identified FcγRIIB/III + (putative DC3) and FcγRIIB/III - (putative DC2) cDC2, can be further separated according to their Clec12A and CD301b expression status ( Figure 5 ). 1 , 12 While this heterogeneity is present within all analyzed tissues, the splenic cDC2 compartment displays a different subset composition. Here, ESAM + cDC2 represent the dominant cDC2 subset.…”

“…The here employed gating strategies were developed and validated via unbiased clustering and a multitude of counterstains for canonical myeloid markers across all tissues. 1 , 2 , 4 , 6 …”

“…Independent on the analyzed tissue, macrophages, plasmacytoid DCs (pDCs), eosinophils, Ly6C high and Ly6C low monocytes as well as cDC1 and cDC2 were identified within the pool of single, alive, CD45 + LIN - cells according to the literature. 1 , 2 , 4 , 7 , 8 , 9 , 10 , 11 Lineage markers included CD3ε, CD19, CD90.2, Ly6G, Ter119, and NK1.1. Further, macrophages, pDCs and eosinophils were gated as MerTK + B220 - , MerTK - B220 + PDCA-1 + Ly6C + , and MerTK - B220 - PDCA-1 - CX3CR1 - CD11b + SSC-A high , respectively.…”

“…Finally, cDC2 were separated into Clec12A + FcγRIIB/III - , Clec12A + FcγRIIB/III + , and Clec12A - FcγRIIB/III - cDC2, corresponding to DC2, DC3 and ESAM + cDC2. 1 Data was collected at a BD LSRFortessa SORP and visualized using FlowJo software (BD Biosciences).

Figure 3 Gating strategy for myeloid cells and pDCs in thymic and skin-draining lymph node-derived single cell suspensions Flow cytometric analysis of single cell suspensions from (A) pooled inguinal, brachial, and axillary skin-draining lymph nodes, and (B) the thymus of C57BL/6J mice.

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“…Finally, cDC2 were separated into Clec12A + FcγRIIB/III - , Clec12A + FcγRIIB/III + , and Clec12A - FcγRIIB/III - cDC2, corresponding to DC2, DC3 and ESAM + cDC2. 1 Data was collected at a BD LSRFortessa SORP and visualized using FlowJo software (BD Biosciences).…”