Thymus tissue was obtained from cardiac surgery patients at the Royal Children’s Hospital (Melbourne, Australia). Our group’s analysis of human NKT cells is part of an ongoing study and, as such, a proportion of the collective thymus and adult blood samples selleck chemical represented

in this study was represented in collective data that formed part of earlier independent studies published by our group. Spleen was obtained from organ donor subjects (Melbourne, Australia). Informed consent was obtained from all donors or their legal guardians. The research was approved by one or more of the Health Sciences Human Ethics Committee (University of Melbourne), the Ethics in Human Research Committee (Royal Children’s

Hospital), the Human Research Ethics Committee (Royal Melbourne Hospital) and the Human Research Ethics Committee (Walter and Eliza Hall Institute of Medical Research). PBMCs were isolated by gradient centrifugation using Histopaque (density 1·077 g/ml; Sigma-Aldrich, St Louis, MO, USA). Thymus tissue was pushed through a stainless steel sieve into complete media (RPMI-1640 medium; Invitrogen Life Technologies, Carlsbad, Aloxistatin manufacturer CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KA, USA), 15 mM HEPES (Invitrogen Life Technologies), 0·1 mM non-essential amino acids (Invitrogen Life Technologies), 100 U/ml penicillin (Invitrogen Life Technologies), 100 μg/ml streptomycin (Invitrogen Life Technologies), 2 mM glutamax (Invitrogen

Life Astemizole Technologies), 1 mM sodium pyruvate (Invitrogen Life Technologies) and 50 μM 2-mercaptoethanol (Sigma-Aldrich). Spleen was digested in RPMI-1640 medium supplemented with 10 mM HEPES, 2 mg/ml collagenase and 0·5 mg/ml DNase at room temperature for 20 min with frequent pipetting; 20 mM ethylenediamine tetraacetic acid (EDTA) was added to stop digestion and undigested fragments were filtered through a stainless steel sieve. Splenocytes were then overlayed on Ficoll and lymphocytes were isolated by gradient centrifugation. PBMCs and splenocytes were usually cryopreserved initially at −80°C [in 10% dimethylsulphoxide (DMSO), 90% fetal bovine serum] before transfer to liquid nitrogen storage. Viability of thawed cells was typically > 90%. NKT cells were isolated from PBMCs by magnetic bead-mediated enrichment and/or fluorescence-activated cell sorting. For magnetic bead enrichment, phycoerythrin (PE)-conjugated, alpha-galactosylceramide (αGalCer)-loaded CD1d tetramer-labelled PBMCs were incubated with anti-PE microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) and passed through an LS column (Miltenyi Biotech) on a MACS Separator (Miltenyi Biotech) according to the manufacturer’s instructions.