Single-cell suspensions of 1 × 106 cells in a 50 μl or 100 μl of whole blood were washed with fluorescence activated cell sorter (FACS) buffer [phosphate-buffered saline (PBS) supplemented with 2% FBS and Vincristine research buy 0·02% sodium azide] and then preincubated with rat anti-mouse CD16/CD32 (clone 2.4G2) to block Fc binding. Specific antibodies were then added to the samples and incubated for 30 min at 4°C. Stained samples were then washed and fixed with 2% paraformaldehyde

for cell suspensions or treated with BD FACS lysing solution for whole blood. At least 50 000 events were acquired on LSRII or FACSCalibur instruments (BD Biosciences). Data analysis was performed with FlowJo (Tree Star, Inc., Ashland, OR, USA) software. Cytokine production by human CD4 and CD8 T cells Angiogenesis inhibitor was quantified using the BD Cytofix/Cytoperm Kit Plus GolgiStop (BD Biosciences), according to the manufacturer’s instructions. Splenocytes were recovered from the indicated mice at 12 weeks after implant of fetal tissues. Red blood cells were lysed and 1 × 106 cells were then left unstimulated or stimulated with phorbol myristate acetate (PMA) (0·5 μg/ml) and ionomycin (0·5 μg/ml) in the presence of GolgiStopTM (0·1 μg/ml) for 4 h at 37°C in 5% CO2. Cells were then fixed and permeabilized using Cytofix/Cytoperm solution and stained with monoclonal antibodies

(mAb) to interferon (IFN)-γ (clone 4S.B3; eBioscience), IL-2 (clone MQ1-17H12; eBioscience), IL-17A (clone eBio64DEC17; eBioscience) and IL-22 (clone IL22JOP; eBioscience). Stained samples were analysed as described above. CD4+ human Treg were identified in the blood of NSG–BLT mice by staining with antibodies specific for human CD25 (clones

MA-251 and 2A3), CD127 (clone A019D5) and forkhead box protein 3 (FoxP3) (clone Chloroambucil 236A/E7). For staining, 100 μl of whole blood were washed with FACS buffer and then preincubated with rat anti-mouse FcR11b. Antibodies specific for human cell surface markers (CD45, CD3, CD4, CD25 and CD127) were added to the samples and incubated for 30 min at 4°C. Stained blood samples were then treated with BD FACS lysing solution for whole blood. Cells were incubated in eBioscience fixation/permeabilization buffer for 60 min and stained with antibodies specific for human FoxP3 in eBioscience permeabilization buffer for 60 min. Stained samples were analysed as described above. Heparinized blood samples from engrafted mice were centrifuged and the plasma was stored at −80°C. Human IgM and IgG levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to the manufacturer’s instructions and an EMax Endpoint ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).