Lymphocyte separation medium

Human peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation with blood buffy coats provided by Wuhan Blood Center. Briefly, buffy coats were diluted 1:1 with phosphate buffer saline (PBS) and carefully layered over the lymphocyte separation medium (Solarbio, China) in 50 ml tube. Tubes were then centrifuged at 800 g with no brake for 20 min at room temperature (RT). PBMCs layer was carefully aspirated and washed with PBS for 3 times for next use.

Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were isolated using lymphocyte separation medium (Beijing Solarbio Science & Technology, Inc., China). NK, CD4⁺ T, T, and CD8⁺ T cells were isolated from PBMCs by negative selection using the human NK, T, CD4⁺T, and CD8⁺T cell isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated cell detected by FCM was up to 95%. CD33⁺ and CD34⁺ cells obtained from BMMCs were isolated using anti-CD33 and anti-CD34 magnetic microspheres, and LS columns according to the manufacturers’ protocols (Miltenyi Biotec GmbH). CD33⁺ and CD34⁺ cells from BMMCs were cultured at 37°C with 5% CO₂ in Iscove’s medium (Invitrogen, Carlsbad, CA, United States) supplemented with 20% fetal bovine serum (Gibco-Invitrogen) and 100 U/mL penicillin and streptomycin (Invitrogen). The partial sample was stored at −80°C for further analysis.

The peripheral blood of six birds (one bird per cage) of each treatment were taken to determine CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ T-lymphocyte percentages by flow cytometry method, as described by Chen et al. (2009) (link). One milliliter anti-clotting peripheral blood was put in a test tube containing 1 mL 0.1 mol/L (pH7.4) phosphate-buffered saline (PBS) and then transferred to centrifuge tube containing 2 mL lymphocyte separation medium (Solarbio, Beijing, China) and centrifuged at 200 × g for 20 min. Approximately 0.5 mL lymphocyte layer was collected, transferred to another centrifuge tube, and then 2 mL PBS added and centrifuged at 200 × g for 5 min. The supernatant was discarded. The cell concentration was determined using the normal counting method of blood cells and then diluted to 1.0 × 10⁶ cells/mL with PBS. The aforementioned 1 mL cell suspension was transferred to another centrifuge tube and centrifuged at 200 × g for 5 min. The supernatant was discarded. The cells were, respectively, stained with 10 μL mouse anti-chicken CD4-phyto-erythrin (BD Pharmingen, United States) and mouse anti-chicken CD8a-FITC (BD Pharmingen) for 15–20 min at RT, and then 2 mL PBS added and centrifugal elutriation performed once. The supernatant was discarded. The cells were resuspended in 0.5 mL PBS and determined by fluorescence-activated cell sorter (BD Pharmingen) (Wu et al., 2012 (link)).

FF samples were carefully collected from follicles with a diameter of ≥18 mm, and centrifuged immediately at 700×g for 5 min. The supernatant was stored at − 80 °C. GCs were obtained by follicular aspiration and isolated from blood cells and cellular debris with a lymphocyte separation medium (Beijing Solarbio Science and Technology Corporation, Beijing, China) by centrifugation at 700×g for 10 min. Residual red blood cells were removed with a red blood cell lysis buffer (Solarbio Science and Technology Corporation, Beijing, China). The GCs were stored at − 80 °C until the time of use. For each patient, the FF and GCs were collected from all follicles and pooled as one sample.