ISOLATION OF HUMAN BLOOD DENDRITIC CELLS BY DISCONTINUOUS NYCODENZ GRADIENT CENTRIFUGATION

The most potent antigen presenting cell present in peripheral blood, l ymphoid 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 identi fication and isolation. Standard techniques for the isolation of brood DC generally employ a period of in vitro culture followed by flotatio n on dense albumin gradients, or more recently, discontinuous gradient s of metrizamide. Dense albumin gradients are time consuming to prepar e, giving low and variable yields of DC. Metrizamide is more convenien t, although exposure of monocytes to metrizamide can decrease the expr ession of CD14 and alter the accessory cell properties of antigen pres enting cells. Here we demonstrate that Nycodenz gradient centrifugatio n of 16 h cultured, T lymphocyte depleted, peripheral blood mononuclea r cells (PBMC) reliably yields a population of low density cells that is highly enriched for DC. Most B and residual T lymphocytes are deple ted and NK cell numbers are reduced two-fold from the interface cell p opulation. The high density pellet fraction exhibits very little allos timulatory 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, CD1 1b, CD14, CD16, CD19 and CD57. Nycodenz exhibits low toxicity, does no t alter the allostimulatory activity of antigen presenting cells, and is therefore ideal for the isolation of cultured DC.