Enrichment of Canine Th17 Cells

Separation of Th17-cells was performed by using a modified protocol by KOL et al. [27 (link)–29 ]. PBMCs were blocked with Human TruStain FcX™ (BioLegend®, California, USA) and subsequently marked with the following antibodies: mouse anti dog CD8 alpha (1:5 diluted with staining buffer), mouse anti dog CD11b (1:11 diluted with staining buffer), mouse anti canine CD21 (first dilution: 1:5 with staining buffer, second dilution: 1:11 with staining buffer) and goat anti-mouse IgG microBeads (1:5 diluted with staining buffer; MACS Miltenyi Biotec, Germany). Afterwards, the cell suspension was separated using the “Deplete” program of autoMACS® Pro Separator (Miltenyi Biotec GmbH, Germany). Thus, undesired cells (CD8 alpha+, CD11b+, CD21+) were sorted out by magnetic columns and the target population of CD3+ and CD4+ cells could be assembled. All antibodies were purchased from Bio-Rad Laboratories, Inc., California, USA.