Anti foxp3 apc

For memory T cell analyses, splenocytes from various vaccinated groups were harvested, lysed with 1x RBC lysis buffer, washed and 1 × 10⁶ cells per well was utilized for each reaction followed by flow cytometry (BD FACSCaliber, Hampton, NH, USA) analysis. Splenocytes were stained with anti-CD44-APC (Biolegend, San Diego, CA, USA), anti-CD4-FITC (Biolegend, San Diego, CA, USA), anti-CD8-PE (Biolegend, San Diego, CA, USA), anti-CD25-APC (Biolegend, San Diego, CA, USA), or anti-Foxp3-APC (Biolegend, San Diego, CA, USA).

We used flow cytometry to verify the proliferation and differentiation of the Th17 cells and Treg cells in the blood. Blood samples were drawn into heparinized tubes and immediately used in the experiment. CD3+CD4+CD8a—IL-17+Th17 cells: cells were stimulated for four hours with Leuko Actvtn Cktl with Glgplg (BD Pharmingen) and RPMI Medium Modified (HyClone) in a constant temperature box at 37°C. Following the instructions of the IntraSureTMKit (BD Pharmingen), RBC Lysis Buffer (CWBIO) was added, and intracellular Foxp3 was stained. CD4+CD25+Foxp3+Treg cells: RBC Lysis Buffer (CWBIO) was added to the blood and washed with PBS, and the surface markers were stained with the corresponding fluorescent-labeled antibodies for 20 min. Intracellular staining of IL-17 was performed using the Transcription Factor Buffer Set (BD Pharmingen). The following antibodies were purchased from BD Pharmingen or Biolegend: anti-CD25-PE, anti-Foxp3-APC, anti-CD4-AF488, anti-CD3-APC, anti-CD8a-PerCP, and anti-IL-17-PE. Flow cytometric data were obtained using a FACSCalibur Flow Cytometer system (BD Pharmingen) and analyzed using FlowJo software.