Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation.
The most potent antigen presenting cell present in peripheral blood, lymphoid and non-lymphoid tissue is the dendritic cell (DC). The study of human DC has been restricted by their low frequency in the tissues and the lack of a truly DC specific surface marker to assist in identification and isolation. Standard techniques for the isolation of blood DC generally employ a period of in vitro culture followed by flotation on dense albumin gradients, or more recently, discontinuous gradients of metrizamide. Dense albumin gradients are time consuming to prepare, giving low and variable yields of DC. Metrizamide is more convenient, although exposure of monocytes to metrizamide can decrease the expression of CD14 and alter the accessory cell properties of antigen presenting cells. Here we demonstrate that Nycodenz gradient centrifugation of 16 h cultured, T lymphocyte depleted, peripheral blood mononuclear cells (PBMC) reliably yields a population of low density cells that is highly enriched for DC. Most B and residual T lymphocytes are depleted and NK cell numbers are reduced two-fold from the interface cell population. The high density pellet fraction exhibits very little allostimulatory activity, indicating that few DC pass into the pellet. The low density fraction contains a significant population (20 +/- 5 (SD)%, n = 8) of cells which fail to stain for the lineage markers CD3, CD11b, CD14, CD16, CD19 and CD57. Nycodenz exhibits low toxicity, does not alter the allostimulatory activity of antigen presenting cells, and is therefore ideal for the isolation of cultured DC.
McLellan
A D
AD
Haematology/Immunology Research Laboratory, Christchurch Hospital, New Zealand.
Starling
G C
GC
Hart
D N
DN
D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D003713:Dendritic Cells / Q000276:immunology / Q000502:physiology; D006801:Humans; D016130:Immunophenotyping; D007472:Iohexol
D007472:Iohexol