Mouse anti chicken cd8α fitc

Monoclonal antibodies against chicken monocyte/macrophages (mouse anti-chicken monocyte/macrophages-PE), CD4 (mouse anti-chicken CD4-PE) and CD8 (mouse anti-chicken CD8α-FITC) were purchased from Southern Biotechnology (Birmingham, Ala, USA). Mouse anti-chicken CD40 and mouse anti-chicken CD86 monoclonal antibodies were used as primary antibodies (purchased from Bio-Rad, Raleigh, NC, USA). Anti-mouse-FITC IgG antibody was used as secondary antibody. Goat anti-mouse IgG, Streptavidin-PerCP/Cy5.5 and Mouse IgG1 isotype control were purchased from Bio Legend (San Diego, CA, USA).

Eighteen hours post-treatment, mononuclear cells were stained with 7-AAD and subsequently fixed for 30 min using the fixation/permeabilization kit (BD Bioscience, Mississauga, ON, Canada). Cells were incubated in fixation and permeabilization buffer for 30 min at 4°C, blocked (20 min at 4°C) with 1% bovine serum albumin (BSA) followed by incubation (20 min at 4°C) with anti-cIFN-γ-biotin (ThermoFisher Scientific, Canada). Cells were washed twice in staining buffer (1% BSA) and stained with streptavidin-APC (ThermoFisher Scientific, Canada) for 20 min at 4°C in staining buffer. Mononuclear cells were washed twice in staining buffer and incubated with a mouse anti-chicken CD3ζ-PB, mouse anti-chicken CD4-PE-CY7 or mouse anti-chicken CD25-PE-CY7, mouse anti-chicken CD8α-FITC, and mouse anti-chicken γδTCR-PE (Southern Biotech, Canada). All cells were acquired on a BD FACS Canto II, and the data were processed by FlowJo V10 software.

The antibodies mouse anti-chicken KULO1-FITC, mouse anti-chicken MHC II-PE, mouse anti-chicken CD3_ζ-PB, mouse anti-chicken CD4-PE-CY7, mouse anti-chicken CD8α-FITC, and mouse anti-chicken γδTCR-PE were purchased from Southern biotech, Canada. The following mouse anti-chicken CD25-APC (Biorad, Canada) and mouse anti-chicken IFN-γ biotin and streptavidin APC were purchased from Life Technologies, Canada.