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.


CONCLUSIONS
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.
Lehner
M
M
Children's Cancer Research Institute, St. Anna Children's Hospital, Vienna, Austria.
Holter
W
W
D002454:Cell Differentiation / Q000276:immunology; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D005362:Ficoll / Q000592:standards*; D005434:Flow Cytometry; D006801:Humans; D018664:Interleukin-12 / Q000032:analysis / Q000096:biosynthesis; D009000:Monocytes / Q000166:cytology* / Q000276:immunology
D018664:Interleukin-12; D005362:Ficoll