Pe conjugated mouse anti chicken cd8α monoclonal antibodies

3 × 10⁵ cells of PBL or tissue single-cell suspensions were simultaneously incubated with APC-conjugated mouse anti-chicken CD3⁺, FITC-conjugated mouse anti-chicken CD4⁺, and PE-conjugated mouse anti-chicken CD8α⁺ monoclonal antibodies (SouthernBiotech, Birmingham, USA) in the dark at 4 °C for 30 min. After three washes with PBS, the labeled cells were analyzed by flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA) within 12 h. The data were analyzed by the software of FlowJo V10 (TreestarInc, Ashland, OR, USA). For the phenotype identification of the CD8⁺ T cell, FITC-conjugated mouse anti-chicken CD8β⁺ and APC-conjugated mouse anti-chicken CD4⁺ monoclonal antibodies (SouthernBiotech, Birmingham, USA) were also used.

A fluorescence-activated cell sorting machine (FACS Aria II, Becton Dickinson, New Jersey, USA) was used to sort a single cell into each well of a 96-well PCR plate containing 2.5μL of 10× Lysis Buffer (Vazyme# N711). Pooled PBMCs were from the mixed equal amount of PBMC samples of three ALV-J infected chickens at 21 DPI. For the isolation of CD8^high+, CD8^medium+, and CD4⁺CD8^low+ (CD8^low+) populations, the pooled PBMCs from ALV-J infected chicken were stained for APC‐conjugated mouse anti‐chicken CD3⁺, FITC‐conjugated mouse anti‐chicken CD4⁺, and PE‐conjugated mouse anti‐chicken CD8α⁺ monoclonal antibodies (Southern Biotech, Birmingham, USA). For CD8^high αα⁺ and CD8^high αβ⁺ population isolation, the pooled PBMCs from ALV-J infected chicken were stained for APC‐conjugated mouse anti‐chicken CD3⁺, PE‐conjugated mouse anti‐chicken CD8α^+, and FITC‐conjugated mouse anti‐chicken CD8β⁺ monoclonal antibodies (Southern Biotech, Birmingham, USA) as described previously (Dai et al., 2020 (link)). Each population of five T lymphocyte subtypes was analyzed in four replications, and each replication sorted 100 single cells for subsequent SMART-Seq2 analysis. Furthermore, the gating strategy for each population was shown in Supplementary Figures 1A, B.