Quantifying Dendritic Cells and Th17 Cells

Single-cell suspensions were initially blocked with an Fc receptor blocker, anti-mouse CD16/32 (Thermo Fisher Scientific, Waltham, MA), on ice for 10 minutes, then stained with fluorochrome-conjugated antibodies for 15 minutes in the dark in accordance with previously established protocols (18 (link), 19 (link)). All flow cytometry analyses were conducted exclusively on the live cell populations, which was achieved by excluding dead cells through staining with Zombie Aqua (BioLegend, San Diego, CA). To quantify type 1 conventional dendritic cells (cDC1) and T helper-17 (Th17) cells, the following anti-mouse primary antibodies were utilized: CD45-APC/Cy7, CD11c-BV421, CD11b-AF700, MHCII-FITC, Ly6C-APC-Cy7, Ly6G-PE, CD3-FITC, CD4-APC, FOXP3-BV421, and RORγT-PE (BioLegend). Activated cDC1 cells were identified as CD45⁺ Ly6G^- MHCII⁺ CD11c⁺ CD11b^- CD86⁺. Th17 cells were identified as CD45⁺ CD3⁺ CD4⁺ RORγT⁺. Analysis was carried out using a BD FACSAria Fusion flow cytometer (BD Biosciences, San Jose, CA), and flow cytometry data were subsequently analyzed using the FlowJo software (FlowJo, Ashland, OR).