Related Experiment Video
Updated: Aug 8, 2026
Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood
Published on: March 17, 2017
A comparative study of immunomagnetic methods used for separation of human natural killer cells from peripheral blood
Anita Øren1, Christina Husebø, Ann-Charlotte Iversen
1Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Medisinsk teknisk senter, N-7489 Trondheim, Norway.
Insights
Comparing immunomagnetic sorting methods for natural killer (NK) cells, Microbeads positive selection offers better yield and activation for NK/NKT cell studies. For pure NK cells, negative sorting is preferred.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Natural Killer (NK) cells are crucial for innate immunity.
- Efficient isolation of NK cells is vital for research and therapeutic applications.
- Existing immunomagnetic sorting methods vary in efficacy.
Purpose of the Study:
- To compare positive and negative immunomagnetic sorting methods for NK cells.
- To evaluate cell composition, yield, and activation.
- To determine the optimal method for different research needs.
Main Methods:
- Comparative analysis of Dynabeads and Microbeads for positive and negative sorting.
- Assessment of cell purity (NK, NKT, monocytes, B cells).
- Evaluation of cell yield, NK cell activation markers (CD69), cytokine production (TNF-α, IFN-γ), and cytotoxicity.
Main Results:
- Microbeads (positive/negative) yielded more cells (14%) than Dynabeads (5%).
- Negative sorting provided better NK cell subpopulation discrimination and CD56 fluorescence.
- Positive Microbeads sorting showed higher cytokine production; Dynabeads showed higher CD69 expression.
Conclusions:
- Positive selection with Microbeads is superior for NK/NKT cell studies due to higher yield and activation.
- Negative sorting is recommended for studies requiring pure NK cells and detailed subpopulation analysis.
- Method choice depends on specific research objectives for NK cell isolation.
Abstract:
Immunomagnetic sorting of natural killer (NK) cells from the peripheral blood of healthy donors has been evaluated in a comparative study of composition, yield and activation of target cells obtained by positive (Dynabeads, Microbeads) and negative (Microbeads) sorting procedures. Positively sorted target cells were selected by expression of the NK cell marker CD56, whereas NK cells obtained by negative sorting were those remaining after steps to remove all non-NK cell leukocyte populations were accomplished. In positive sorting, both CD56+CD3- NK cells and CD56+CD3+ natural killer T (NKT) cells were included. The NKT cell fraction differed between individuals, but not between the positive sorting methods. Whereas 20-30% of positively sorted target cells were NKT cells, only approximately 3% of negatively sorted cells were CD3+. Contamination with monocytes and B cells was low (1-3%) in all methods studied. Sorting with Microbeads (both positive and negative) gave higher cell yields than those obtained with Dynabeads (14% vs. 5% of total leukocyte numbers). A higher CD56 fluorescence intensity of NK cells and a better discrimination between the CD56bright and CD56dim NK cell subpopulations was obtained after negative sorting. Dynabeads-separated cells had, shortly after separation, a significantly higher expression (approximately 30%) of the early activation marker CD69 than cells either positively or negatively separated by Microbeads (approximately 8%). CD56+ cells positively sorted by Microbeads demonstrated a significantly higher production of TNF-alpha and IFN-gamma after IL-2 stimulation than Dynabeads-sorted cells. However, the cytotoxicity of cells obtained by the two positive sorting procedures did not differ. In conclusion, positive selection of CD56+ cells by Microbeads is better than Dynabeads, as determined from cell yield and procedure-associated cell activation and should be chosen for in vitro studies of NK/NKT cells. However, when pure NK cells and phenotypic subtypes are to be studied, negative sorting seems most appropriate.
