Pe pdca1

The spleen was dissociated into a single‐cell suspension using PBS supplemented with 10% fetal bovine serum in GentleMACs tubes (Miltenyi Biotec). Red blood cells were lysed by resuspending splenic cells in ammonium chloride‐Tris buffer and incubation at room temperature for 8 minutes. Leukocytes were collected following centrifugation and washed twice in PBS. The splenic leukocytes were resuspended in PBS and enriched to 2×10⁶/μL. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher) was used to check the viability of splenic leukocytes. Specimens (100 μL each) were treated with CD16/32 blocking antibodies for 10 minutes followed by antibody mixtures (APC/Cy7‐CD45, FITC‐CD3, PerCP/Cy5.5‐B220, APC‐IA/IE, PE‐PDCA1, BV421‐CD11c) (BioLegend, San Diego CA) for 30 minutes. Corresponding fluorescence minus one controls were also prepared. Then, all specimens were fixed by adding 500 μL 4% paraformaldehyde (Sigma‐Aldrich). Flow cytometry was performed using Attune NxT Flow Cytometer (ThermoFisher), and data were analyzed using FlowJo software (BD Company, Ashland, OR). The pDCs were defined by CD45⁺, CD3‐/IA/IE⁺ and B220+/PDCA1⁺, and cDCs by CD45⁺ and CD11c+/IA/IE⁺.