Related Experiment Video
Updated: Aug 11, 2026
An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets
Published on: April 18, 2016
Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation
A D McLellan1, G C Starling, D N Hart
1Haematology/Immunology Research Laboratory, Christchurch Hospital, New Zealand.
Insights
Researchers developed a new method using Nycodenz gradient centrifugation to isolate dendritic cells (DCs) from peripheral blood mononuclear cells (PBMC). This technique offers a more reliable and less toxic way to obtain highly enriched DC populations for research.
Area of Science:
- Immunology
- Cell Biology
Background:
- Dendritic cells (DCs) are crucial antigen-presenting cells but are difficult to isolate due to low frequency and lack of specific markers.
- Existing isolation methods like albumin or metrizamide gradients have drawbacks, including time consumption, low yields, or altered cell properties.
Purpose of the Study:
- To establish a reliable and efficient method for isolating dendritic cells (DCs) from human peripheral blood mononuclear cells (PBMC).
- To evaluate Nycodenz gradient centrifugation as a superior alternative to existing DC isolation techniques.
Main Methods:
- Peripheral blood mononuclear cells (PBMC) were cultured for 16 hours and depleted of T lymphocytes.
- Nycodenz gradient centrifugation was used to separate low-density cells enriched for DCs.
- Flow cytometry was employed to analyze cell populations and lineage marker expression.
Main Results:
- Nycodenz gradient centrifugation reliably yielded a low-density cell population highly enriched for dendritic cells (DCs).
- This method effectively depleted B and T lymphocytes and reduced NK cell numbers.
- The isolated low-density fraction contained a significant population of cells lacking common lineage markers, indicating high DC purity.
- Nycodenz demonstrated low toxicity and preserved the allostimulatory capacity of antigen-presenting cells.
Conclusions:
- Nycodenz gradient centrifugation is an effective and non-toxic method for isolating cultured dendritic cells (DCs) from PBMC.
- This technique overcomes limitations of previous methods, providing a valuable tool for immunological research.
Abstract:
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.
