Apc conjugated mouse anti human cd11c

Monocytes were purified from buffy coats of healthy donors (Karolinska University Hospital) by using a RosetteSep monocyte purification kit (Stem Cell Technologies) and Ficoll-Hypaque Plus (Amersham Biosciences) gradient centrifugation. Human DCs were seeded at 0.8 × 10⁶ to 1.5 × 10⁶ cells/ml in R10 (RPMI 1640, 2 mM l-glutamine, 10% fetal bovine serum [FBS]) supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF; 40 ng/ml) and IL-4 (40 ng/ml) (both from PeproTech) for 6 days. Cells were given fresh media and cytokines at a ratio of 1:1 on day 4 and cultured until day 6. The human DC phenotype was assessed by examination of CD11c and CD1a expression via staining with allophycocyanin (APC)-conjugated mouse anti-human CD11c and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD1a (BD Pharmingen). DCs used in these experiments were above 90% CD1a⁺/CD11c⁺.

Buffy coats from healthy donors were obtained from the blood bank at the Karolinska University Hospital (Immunology/Transfusion Medicine Clinic, http://www.karolinska.se/for-vardgivare/kliniker-och-enheter-a-o/kliniker-och-enheter-a-o/karolinska-universitetslaboratoriet/klinisk-immunologitransfusionsmedicin/bestallning-av-blodkomponenter-for-fou/). The buffy coats were provided anonymously; hence informed consent was not required. Monocytes were isolated using RossetteSep^™ human monocyte enrichment kit (StemCell Technologies) and Ficoll-Paque PLUS (GE healthcare) according to the manufacturer′s instructions. To obtain DCs, the cells were then cultivated in RPMI 1640 medium (+L-glutamine) supplemented with 10% heat inactivated fetal bovine serum (FBS) (from Invitrogen) in the presence of 50 ng/ml human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml human recombinant interleukin (IL)-4 (PeproTech). On day 3, cells were given fresh media and cytokines (ratio 1:1) and cultured until day 6. The DC phenotype was assessed by examination of CD11c and CD1a expression using allophycocyanin (APC) conjugated mouse anti-human CD11c and fluorescein isothiocyanate (FITC) conjugated mouse anti-human CD1a (BD Pharmingen) before use.