Lymphocyte separating medium

The following details concerning the antibodies (Abs) and reagents were used in this study: mouse anti-chicken [MHC-class II⁺ (clone-2G11), CD86⁺ (Clone-GL1), CD1.1⁺ (Clone-CB3), CD11c⁺ (Clone-N418)] with appropriate isotype IgG1-PE, IgG2a-PE/Cy7, IgG1-FITC, IgG-APC, respectively, all were from Southern Biotech (Birmingham, AL). Hiscript II Q RT SuperMix for QRT-PCR (⁺gDNA Wiper) (L/N: 7E312C9) and ChamQ SYBR QRT-PCR Master Mix are from Vanzyme. TRIzol (L/N: A304-1) from TaKaRa Biotechnology (Dalian) Co. Ltd., Dalian City, Liaoning, China. The enhanced horseradish peroxidase (HRP)-DAB chromogenic Kit (L/N: S7418) was from TIANGEN Co. Ltd. (Tiangen, China). Freund's adjuvant Complete and Freund's adjuvant Incomplete were from (Sigma Aldrich, Merck KGaA, Darmstadt, Germany). Lymphocyte separating medium (L/N: LTS1077) was from TBD-science, Tianjin, China. Gibco RPMI Medium-1640 (L/N: 2037577) and Chicken Serum was from Life Technologies, San Francisco, CA. Recombinant chicken-granulocyte macrophage colony-stimulating factor (GM-CSF) was from Abcam, Cambridge, UK, and recombinant chicken interleukin 4 (IL-4) was from Kingfisher, London, UK.

Spleens of 3 three-week-old birds were collected and isolated from a thin surrounding layer using sterile instruments and placed into a Petri plate with enough sterile PBS. Single-cell suspensions were prepared from spleens by gentle mashing through the nylon mesh filter and mixed with sterile PBS 1:1. Splenocytes were obtained after loading onto an equal volume of cell suspension and lymphocyte separating medium (TBD-science, Tianjin, China) and centrifuging at 600 × g for 20 min. Trypan blue negative cells were counted as viable under a microscope in a hemo-cytometer chamber, and approximately 2.5 × 10⁶ cells were obtained from each bird.

Blood samples were collected from humans and mice in ethylenediaminetetraacetic acid (EDTA) tubes, and PBMCs were isolated within 4 hours. The supernatant was removed after centrifugation at 1500 rpm for 10 minutes. The precipitate was resuspended in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) and gently added to the surface of 3 mL of lymphocyte‐separating medium (TBD Science, China). The samples were centrifuged at 2000 rpm for 20 minutes and then gently collected and washed twice with DMEM to remove debris. Cells were resuspended in TRIzol reagent (Thermo Fisher Scientific, USA) and stored at −80°C for further analysis.