Cell isolation, purification, and FACS analisys
Sodium-heparinized peripheral venous blood samples were kindly provided by the Immunohematology and Transfusion Medicine Service of San Raffaele hospital, Milan, Italy and were derived from healthy donors who donate blood. Highly purified (98%) naive Treg were isolated using the FACS Aria II according to the expression of CD4-PB (Clone SK3), CD25-APC-Cy7 clone (eBioDDR5), CD127-PE-CY7 (clone HIL-7R-M21), CD45RA-PE-Cy5 (clone HI-100), CD62L-PE (clone DREG-56), and CD95-APC (clone DX2) all from BD Biosciences (Supplemental figure 1). The same antibodies were used for FACS analysis and with the additional staining for FOXP3-AF488 (clone PCH101, from eBioscience). According to the experimental needs STAT-5 phosphorylation was measured using anti STAT5 pY694 Alexa Fluor 647 (clone 47, BD Biosciences) and Bcl-2 was detected using anti Bcl-2 APC (clone Bcl-2/100, BD Biosciences). Telomere length was measured using a fluorescein conjugated PNA probe kit from DAKO. Apoptosis was measured using an annexin V- propidium iodide staining kit from BD Biosciences.