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Two multi-color immunophenotyping panels for assessing the innate and adaptive immune cells in the mouse mammary gland

Ashleigh Unsworth, Robin Anderson, Nicole Haynes, Kara Britt Cytometry PART A, Volume 89, Issue 6, 527–530 (2016)

PURPOSE: A multi-color antibody panel was designed and optimized to identify and characterize 10 leukocyte subpopulations from both the innate and adaptive arms of the immune system. Markers to detect B-cells (CD45.2+ CD19+), T-cells (CD45.2+ TCRβ+), natural killer cells (CD45.2+ TCRβ− CD49b+ NKp46+), and myeloid cells (CD45.2+ CD11b+) were employed. Myeloid cells were further differentiated as dendritic cells (CD45.2+ CD11b+ CD11c+ MHCII+), neutrophils (CD45.2+ CD11b+ Ly6G+), macrophages (CD45.2+ CD11b+ Ly6G− Ly6Clow), and monocytes (CD45.2+ CD11b+ Ly6G− Ly6Chigh). T-cell populations were sub-divided into T-helper cells (CD45.2+ TCRβ+ CD8− CD4+), and cytotoxic T-cells (CD45.2+ TCRβ+ CD4− CD8+). T-cell memory/effector status was determined using CD62L and CD44 to distinguish between effector (CD44+ CD62L−), memory (CD44+ CD62L+) and naïve (CD44− CD62L+) T cells. This panel was established for the analysis of collagenase digested mouse (Balb/C) mammary gland, spleen and tumor samples, as well as RBC-lysed whole blood (Table 1).

CELL TYPE: RBC-lysed whole blood, Collagenase IV digested spleen, Collagenase IV digested mammary gland, Collagenase IV digested tumor

MACHINE: BD LSR Fortessa X20

Marker Target Clone Fluorochrome Purpose
CD45.2 Mouse 104 APC-Cy7 Leukocyte
CD11c Mouse HL3 APC DC
Ly-6G Mouse 1A8 PE-Cy7 Neutrophil
CD11b Mouse M1/70 PE Myeloid cell
MHC II Mouse M5/114.15.2 Brilliant Violet 711 DC
Ly-6C Mouse AL-21 Brilliant Violet 421 Monocyte, macrophage
Mannose Receptor Mouse C068C2 FITC alternatively activated macrophage
CD4 Mouse Gk1.5 APC-Cy7 Th
TCR beta Mouse H57-597 PE-Cy7 T cell
CD45.2 Mouse 104 PE leukocyte
CD49b Mouse DX5 FITC NK
CD8 Mouse 53-6.7 Brilliant Violet 711 cytotoxic T cell
CD44 Mouse IM7 Brilliant Violet 605 T cell differentiation
CD62L Mouse Mel-14 Brilliant Violet 510 T cell differentiation
CD19 Mouse 1D3 APC B cell
NKp46 Mouse 29A1.4 Brilliant Violet 421 NK
Dead cells Mouse PI Viability
Dead cells Mouse Fluoro-Gold Viability