Ficol hypaque

Human peripheral blood cell samples were obtained from healthy donors (Transfusion medicine department, Clinical center, National institutes of health, Bethesda, MD, with an approved human subject agreement) by leukopheresis. Peripheral blood mononuclear cells (PBMC) were isolated by Ficol-Hypaque (Sigma-Aldrich, St-Louis, MO) density gradient centrifugation. Monocytes and CD4+ T cells were purified (>95%) from PBMC with the use of MACS CD14 monocyte and CD4 T isolation kits (Miltenyl Biotech, Auburn, CA) according to the manufacturer instructions (4 (link), 5 (link)). Mouse bone marrow derived hematopoietic progenitor cells (HPCs) were prepared from C57BL/6 wild type mice (Male 7–8 weeks old) by flushing from femur and tibia with the depletion of red blood cell (RBCs) by ammonium chloride treatment [ACK lysing buffer, Quality Biological] (18 (link)).

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficol hypaque (Sigma-Aldrich, St. Louis, USA) gradient centrifugation and suspended in RPMI-1640 (Caisson Laboratories, USA) supplemented with L-Glutamine (Sigma, USA), HEPES (Sigma, USA), antibiotics (Biological Industries, Israel) and 10% heat inactivated fetal calf serum (FCS) (Biological Industries, Israel) as per previously published protocol from our laborartory [9 (link)]. PBMCs were cultured (2x10⁶ cells/ml) with either PMA+Ionomycin (as positive control) for 6 hours or M. leprae antigen (WCL, 20μg/ml) for 48 hours at 5% CO₂, 37°C and 1μM Monensin (Sigma, USA) added for last 6 hours. At the end of culture period, cells were stained for intracellular cytokines IL-10, IL-17, IFN-γ and the transcription factor FoxP3 and cell surface markers CD4, CD25, CD45RO, PD-1, PDL-1, CCR4 and CCR6. The cells were finally suspended in staining buffer and acquired on BD FACS Calibur; USA and analysis performed on Flowjo software.