2.5 Flow
cytometry
At indicated time points, whole blood and single cell suspensions of
spleen, ghLN, lung, and liver were stained for flow cytometric
analyses. 50 µl whole blood and 50 µl single cell suspensions
(approx. 1 × 106 leukocytes) were used for staining.
To identify iNKT cells, whole blood was incubated with PBS57-loaded
murine CD1d (mCD1d) tetramers at room temperature for 30 min in the dark
as described previously . All further incubation steps with monoclonal
antibodies (mAbs) targeting extracellular antigens were carried out for
15 min at 4°C in the dark. Between each antibody staining, a washing
step was performed. Before intracellular labeling, erythrocytes in blood
samples were lysed with red blood cell lysis buffer (1.55 M
NH4Cl, 100 mM KHCO3, 12.7 mM
Na4EDTA, pH 7.4, in Aqua destillata ).
Subsequently, samples were fixed and permeabilized with the True-Nuclear
Transcription Factor Buffer Set (Biolegend, USA) according to the
manufacturer’s instructions. All incubation steps for intracellular
staining were carried out for 30 min at 4°C in the dark. Antibodies and
conjugates used for flow cytometry are shown in table 2. The mCD1d
tetramer was obtained from the NIH Tetramer Core Facility.
Dead cells were excluded by FSC/SSC characteristics and using Zombie
Aqua (Biolegend, USA). Single cells were identified by consecutive FSC‑W
vs. FSC‑H and SSC‑W vs. SSC‑H gating. Live, single lymphocytes were
further divided into CD3+/γδ T cell receptor
(TCR)– (αβ T cells), CD3+/γδ
TCR+ (γδ T cells), and CD3+/mCD1d
tetramer+ (iNKT cells). Further subpopulations were
gated according to the markers described in the figures and text. At
least 1 × 104 single, live αβ T cells were recorded.
CD4 was not detectable in one of the control wild boar in trial 2,
presumably because of a polymorphism in the CD4 alleles .
CD4+ and
CD4+/CD8α+ cells from this animal
were therefore not included in the analyses.
Flow Cytometer BD FACS Canto II with FACS DIVA Software (BD Bioscience,
San Jos, CA) and FlowJoTM V10 for Windows (Becton,
Dickinson and Company; 2019) were used for all analyses.