Mouse igg1 isotype control

PBMCs from specific pathogen-free cats (one 3 year olds, four 4 year old and two 6 year old), which are kept in our veterinary teaching hospital or NIPPON ZENYAKU KOGYO Co., Ltd. as a blood donor, were separated by density gradient centrifugation using Lymphoprep (Axis-shield, Oslo, Norway). For PD-1 and PD-L1 expression analysis, the isolated PBMCs were cultured in R10 for 1.5 h to attach monocytes onto the culture dish, and only the lymphocytes in the supernatant were collected. The collected PBLs were cultured in R10 medium in the absence (unstimulated control) or presence of 10 µg/mL of Con A at 37 °C for 48 h. The cells were stained with antibodies for flow cytometric analysis.
For the IFN-γ production assay, PBLs are collected by incubating PBMCs and attaching monocytes as in the flow cytometry analysis, 2 × 10⁵ PBLs of were cultured in R10 medium in the presence of 10 µg/mL of Con A with 10 µg/mL of mouse IgG₁ isotype control (Biolegend) or anti-PD-1 antibody (1A1-2), or rat IgG_2a isotype control (Biolegend) or anti-PD-L1 antibody (G11-6), or cat IgG isotype control (Jackson ImmunoResearch Laboratories) or anti-PD-1 chimeric antibody (ch-1A1-2) at 37 °C for 48 h. The cell supernatants were collected, and the amount of IFN-γ was measured with DuoSet ELISA Development System feline IFN-γ (R&D systems, Inc.).

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).