Endotoxin-free purification of monocytes for dendritic cell generation via discontinuous density gradient centrifugation based on diluted Ficoll-Paque Plus.

BACKGROUND: For the investigation of the biology of human monocyte-derived dendritic cells (DCs), and also for further clinical use of these cells, it is important to separate large numbers of monocytes without isolation-induced activation.
METHODS: Monocytes were purified from mononuclear cells (MNCs) by density gradient centrifugation using Ficoll-Paque Plus adjusted to a density of 1,068 g/ml by dilution with phosphate-buffered saline (PBS) and compared to Percoll isolated monocytes. DCs were generated by culturing monocytes with interleukin (IL)-4 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), and they were functionally tested for IL-12 p70 production by stimulation with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma).
RESULTS: Density gradient centrifugation using diluted Ficoll-Paque Plus resulted in a monocyte purity of more than 75% and a recovery of 70%. DCs derived from such monocytes produced high amounts of IL-12 p70 in response to LPS plus IFN-gamma, thus demonstrating an important functional capacity.
CONCLUSION: Since Ficoll-Paque Plus is commercially available in an endotoxin-free form, this modified Ficoll gradient offers a simple modality for isolating large amounts of monocytes under endotoxin-free conditions without introducing further potential monocyte-activating agents or procedures. Copyright 2002 S. Karger AG, Basel