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OMIP-061

20‐Color Flow Cytometry Panel for High‐Dimensional Characterization of Murine Antigen‐Presenting Cells

Anthony T. DiPiazza, Juliane P. Hill, Barney S. Graham, Tracy J. Ruckwardt Cytometry PART A, Volume 95, Issue 12, 1226-1230 (2019)

PURPOSE: This 20‐color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen‐presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I‐A/I‐E, CD24, CD64, pDCA‐1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co‐stimulation (CD80), and adhesion (ICAM‐1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen‐bearing cells in vivo.

CELL TYPE: B cells, neutrophils, monocytes, macrophages, dendritic cells

MACHINE: BD FACSymphony A5

Marker Target Clone Fluorochrome Purpose
Dead cells Mouse Live/Dead Fix Blue Viability
CD45R (B220) Mouse RA3-6B2 BUV496 B cells and pDCs
CD172a Mouse P84 PerCP-eFluor 710 cDC2, other myeloid cells
CD11c Mouse N418 Brilliant Violet 421 macrophage and DC
CD103 Mouse M290 Brilliant Violet 510 Lung migratory cDC1
Ly-6C Mouse HK1.4 Brilliant Violet 570 Monocytes/macrophages, neutrophils, moDC
CD24 Mouse M1/69 Brilliant Violet 605 cDC lineage
XCR1 Mouse ZET Brilliant Violet 650 pDC (plasmacytoid DC)
MHC Class II (I-A/I-E) Mouse M5/114 Brilliant Violet 711 Activation, antigen p
CD3 Mouse 17A2 Brilliant Violet 750 T and NKT cells
CD64 Mouse X54-5/7.1 Brilliant Violet 786 Monocyte/macrophage, moDC lineage
ccr7 Mouse 4B12 PE Trafficking
CD80 Mouse 16-10A1 PE-CF594 Co-stimulation
F4/80 Mouse BM8 PE-Cy5 Macrophage subsets
CD169 Mouse 3D6.112 PE-Cy7 Macrophage subsets
ICAM-1 Mouse BUV395 Adhesion
CD45 Mouse 30-F11 BUV661 Hematopoietic cell lineage
Ly-6G Mouse 1A8 BUV737 Neutrophil
CD317 Mouse 927 APC pDC
CD11b Mouse M1/70 APC-R700 Macrophages and dendritic cells