Il 4 pe

Cryopreserved PBMCs were recovered for FACS analysis and were pretreated by blocking in blocking buffer (1% FBS/PBS) with Human TruStain FcX Fc receptor blocking solution (BioLegend) per 1,000,000 cells. Cells were stained with the following fluorochrome-tagged antibodies, which have been validated to be specific or cross-reactive to canine immune cell epitopes, using manufacturer’s recommendations: (1) T and B cell panel: CD44-FITC (BioRad, YKIX337.78), CD5-PE (BioRad, YKIX322.3), CD79α-PerCP Cy5.5 (Invitrogen, clone HM47), CD4-PE Cy7 (BioRad, YKIX302.9), CD11b-PE CF594 (BioLegend, clone M1/70)^{[27 (link),28 ]}, CD21-AF647 (BioRad, CA2.1D6), CD45RA (BioRad, clone CA4.1D3), goat anti-mouse IgG APC Cy7 (BioLegend), CD8α AF700 (BioRad, clone YCATE55.9), CCR7 (BD, clone 150503)^{[29 (link)]}, C45RA (BioRad, clone CA4.1D3), and Live/Dead Aqua (Invitrogen); and (2) T cell function panel: CD107β-FITC (BioRad, clone AC17), IL-4 PE (BioRad, clone CC02), CD4-PE Cy7 (BioRad, clone YKIX302.9), TNFα-PE CF594 (BioLegend, clone MaB11)^{[30 (link)]}, IFNγ-AF647 (BioRad, clone CC302), CD8α-AF700 (BioRad, clone YCATE55.9), Granzyme B-Pacific Blue (BioLegend, clone GB11)^{[31 (link)]}, and Live/Dead Aqua (Invitrogen). Cells were acquired on an LSR II. Flow cytometry gating was determined using cells stained with secondary only & single-color controls, and data were analyzed using FlowJo 10.