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Fig. 2 | BMC Veterinary Research

Fig. 2

From: Approaches to overcome flow cytometry limitations in the analysis of cells from veterinary relevant species

Fig. 2

Staining and gating strategy to identify leukocyte populations in ovine blood. Peripheral blood mononuclear cells were obtained by density gradient centrifugation and (a) cells were gated on FSC-Height and FSC-Area to exclude doublets. Singlets were further gated on SSC-Area and LIVE/DEAD™-dyelow cells to exclude dead cells. Viable cells were then gated for lymphocytes and granulocytes based on FSC and SSC properties. Lymphocytes were gated for γδ T cells (a), CD8+ T cells (b), CD4+ T cells (c), and B cells (d). Granulocytes were gated for CD11c+ antigen presenting cells (E). (b) Each leukocyte subset (A-E) was analyzed for expression of α4- and β1-integrins (top row) and CD21 and CD62L (bottom). (c) Fluorescence-minus-one controls (FMO) for fluorochromes used to stain α4- and β1-integrins, CD21, and L-selectin in (b) using the total lymphocyte gate shown in Panel (a). (d) Table indicating the reagents and flow cytometer configuration used in this staining panel, including the respective fluorochrome, as well as the methods employed to visualize each ovine cell surface marker. Reagents that were obtained as covalently labeled reagents are marked as “directly conjugated”. (a and b) Numbers within dot plots represent percentages. Abbreviations: AF™, Alexa Fluor™; Bio, biotin; BV, Brilliant Violet™; FITC, fluorescein isothiocyanate; LP, long pass; mAb, Monoclonal antibody; PE, R-phycoerythrin; PE-Cy7, phycoerythrin-cyanine7 conjugate; PerCP-Cy5.5, peridinin chlorophyll-A protein-cyanine; SA, streptavidin

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