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EditedEdited byby
KerryKerry . CampbellCampbell
arco ColonnaColonna
HUMANA PRESS
Cloning Human NK Cells 1
1
Cloning Human Natural Killer Cells
Marina Cella and Marco Colonna
1. Introduction
Our understanding of the phenotypical and functional heterogeneity of
human natural killer (NK) cells has greatly advanced over the past few years.
This advancement has been greatly helped by the development of culture con-
ditions for clonal proliferation of NK cells. Analysis of human NK cell clones
has led to the original observation that different NK cell clones recognize distinct
major plex (MHC) class I specificities. This has prompted
the production of monoclonal antibodies directed to NK cell surface antigens
clonally distributed, and, ultimately, the biochemical and molecular definition of
the NK cell surface glycoproteins functioning as MHC class I receptors.
Here we describe a limiting dilution culture protocol that allows establish-
ment of human NK cell clones from peripheral blood leukocytes with high
efficiency.
2. Materials
1. RPMI 1640 (Gibco, cat. no. 31870-025).
2. RPMI + HEPES (25 mM): (Gibco, cat. no. 42401-018).
3. Lymphocyte separation medium (LSM): (ICN Biomedicals, cat. no. 50494/36427).
4. Human binant interleukin (IL)-2 Liquemin (25000 UI/5 mL, Roche).
5. RPMI 8866 cells (available from Dr. Bice Perussia, Jefferson Medical College, Kimmel
Cancer Institute, BLSB 750, 233 S 10th Street, Philadelphia, PA 19107, USA).
6. Antibodies: Anti-human CD56 (Pharmingen, cat. no. 31661A, mouse IgG1), anti-
human CD3 (OKT3, mouse IgG2a, , cat. no. CRL-8001), anti-mouse IgG1-
phycoerythrin (PE; SBA, cat. no. 1070-09), anti-mouse IgG2a-fluorescein
isothiocyanate (FITC; SBA, cat. no. 1080-02).
7. Phosphate-buffered saline (PBS).
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