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OMIP-065

Dog Immunophenotyping and T‐Cell Activity Evaluation with a 14‐Color Panel

Stanislav Pantelyushin, Elisabeth Ranninger, Regula Bettschart‐Wolfensberger, Johannes vom Berg Cytometry PART A, Volume 97, Issue 10, 1024-1027 (2020)

PURPOSE: The purpose of the panel described here is to assess the immune cell composition and their functionality in the peripheral blood mononuclear cells (PBMCs) of dogs. Moreover, its “plug and play” composition allows for an in‐depth analysis of T‐cell responses in ex vivo assays (Table 1). Initially, this panel has been designed for the analysis of cryopreserved PBMCs to allow batched analysis and to reduce interexperimental variation. Withers and colleagues published a comparable and—to our knowledge—currently the most extensive canine panel to date (1). While their study focused on the aging and activation status of T cells in dogs, our panel is designed to look at a broader range of cells with a higher number of markers. This allows a more in‐depth analysis of functional extracellular and intracellular markers. In addition, all antibodies in our proposed panel are directly labeled. In combination with suitable lymphocyte isolation protocols, this panel could potentially also be adapted to analyze tissue biopsies from various different organs.

CELL TYPE: PBMC

MACHINE: BD LSR Fortessa

Marker Target Clone Fluorochrome Purpose
Dead cells Dog Zombie Aqua viability
CD45 Dog YKIX716.13 eFluor 450 Leukocyte
CD25 Dog P4A10 SuperBright 600 Treg, activation
CD4 Dog YKIX302.9 SuperBright 645 CD4+ T
CD8a Dog YCATE55.9 SuperBright 702 CD8+ T
CD14 Dog M5E2 Brilliant Violet 785 Monocyte
CD3 Dog CA17.2A12 FITC T
EOMES Dog WD1928 PerCP-eFluor 710 CD8+ T transcription factor
CD22 Dog RFB-4 PE B
Granzyme B Dog GB11 PE-CF594 cytotoxic activation, NK
FoxP3 Dog FJK-16s PE-Cy7 Treg
MHC II Dog YKIX334.2 APC APC
Ki-67 Dog SolA15 Alexa Fluor 700 proliferation
CD5 Dog YKIX322.3 APC-eFluor 780 T, NK