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.