Anti hcd25 apc

The study protocol was approved by The Ethics Committee of The First Affiliated Hospital of Sun Yat-Sen University and used for research purposes only (Approval No. 2012-349). Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors by Ficoll density gradient centrifugation. CD14⁺ cells were isolated from PBMCs using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured with or without 20% TSN for 6 days to obtain macrophages.
To isolate CD4⁺CD25⁻ T cells and Treg subsets, cells were stained with anti-hCD4-FITC, anti-hCD25-APC, and anti-hCD45RA-eFluor 450 (eBioscience, San Diego, CA, USA) antibodies, and sorted using a BD Influx cell sorter (BD Biosciences). Three Treg subsets were prepared as live cells as described previously (Miyara et al, 2009 (link)). Foxp3^lowCD45RA⁺ cells, which were CD25⁺⁺ (I), Foxp3^highCD45RA⁻ cells, which were CD25⁺⁺⁺ (II), and Foxp3^lowCD45RA⁻ cells, which were CD25⁺⁺ (III), were prepared by sorting CD25⁺⁺CD45RA⁺, CD25⁺⁺⁺CD45RA⁻, and CD25⁺⁺CD45RA⁻ cells, respectively.