Cd69 pe

FACS analysis was performed using standard methodology. Briefly, after Fc blocking, 1 × 10⁶ cells were stained with appropriate antibodies for 30 min at 4°C and then washed with PBS/2% FBS. The cells were stained in FACS buffer (PBS containing 0.5% bovine serum albumin and 0.05% sodium azide) with fluorescently labelled antibodies from BioLegend (San Diego, CA; CD11c-APC/Cy7, CD11b-BV421, CD40-FITC, CD80-APC, CD86-PE, CD8-PE/Cy7, CD4-PerCp/Cy5.5, CD69-PE, CD25-APC, CD3-BV421, CD44-BD427, CD45.1-FITC, CD3-PerCp/Cy5.5, and CD44-FITC) or Tonbo Biosciences (San Diego, CA; HLA-DR-PerCp/Cy5.5) to identify DC and T cells. The percentage for each marker was determined by flow cytometry on a Sony SH800 cell sorter, and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). Fluorescence minus one (FMO) control for each marker was used to identify gating boundaries. DC responses were examined by flow cytometry after staining with anti-CD11c, MHCII, CD8, CD4, CD11b, GR-1, CD103, CD80, and CD86 antibodies. TipDC were sorted using CD11b^hi, Ly6c/GR-1^hi, MHCII ^hi DC surface marker staining on a FACS Aria cell sorter (BD Biosciences, Franklin Lakes, NJ).