Isolation and Characterization of Human Natural Killer Cell Subsets

Megan A. Cooper1, Michael A. Caligiuri1

1 The Ohio State University Comprehensive Cancer Center, Columbus, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.34
DOI:  10.1002/0471142735.im0734s60
Online Posting Date:  May, 2004
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Natural killer (NK) cells are innate immune lymphocytes that play a critical role in the host defense against pathogens through their production of cytokines and cytotoxic activity. Human NK cells can be divided into two subsets, each with distinct phenotypic and functional properties, based on cell‐surface density expression of CD56 (CD56bright and CD56dim). This unit describes the purification of human NK cell subsets from blood, protocols for the further characterization of NK cell function, and further background information on these cell types.

Keywords: lymphocyte; human natural killer (NK) cell; CD56; innate immunity

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Table of Contents

  • Basic Protocol 1: Isolation of Human NK Cells from PBMC
  • Alternate Protocol 1: Enrichment of NK Cells
  • Support Protocol 1: Titration of Fluorescently Conjugated Monoclonal Antibodies
  • Support Protocol 2: Phenotypic and Functional Analysis of Human NK Cell Subsets
  • Support Protocol 3: Labeling P815 Cells for NK Cell ADCC Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation of Human NK Cells from PBMC

  • Sodium heparin
  • Human peripheral blood obtained as a leukopac (available from the American Red Cross or local blood banks) or obtained via venipuncture or leukapheresis ( appendix 3F)
  • RosetteSep antibody (human NK cell enrichment) cocktail (StemCell Technologies)
  • PBS ( appendix 2A)
  • RPMI‐1640 medium, 4°C
  • RPMI‐10HAB (see recipe), freshly filtered with 0.2‐µm culture filter (4°C/room temperature)
  • Red blood cell (RBC) lysis buffer (see recipe), room temperature
  • Mouse IgG fluorescently‐conjugated isotype control antibody (Coulter)
  • Anti‐human CD56 fluorescently‐conjugated antibody (Ab) (Coulter, NKH‐1 clone, preferably PE‐ or PC5‐conjugated)
  • RPMI‐30HAB (see recipe), freshly filtered with 0.2‐µm culture filter (4°C)
  • 50‐ml sterile, conical screw‐top polypropylene centrifuge tubes
  • Tabletop rotator (Adams Nutator, Fisher Scientific)
  • 6‐well cell culture dish, optional
  • 37°C, 5% CO 2 humidified incubator
  • Benchtop swinging bucket refrigerated centrifuge (Beckman Allegra 6 with a GH 3.8 rotor or similar)
  • 5‐ml sterile polypropylene or polystyrene flow cytometry tubes with caps (Fisher Scientific)
  • Sterile cell strainer (30‐ to 70‐µm, Fisher Scientific)
  • Flow cytometer capable of cell sorting (Coulter EPICS series, Becton‐Dickinson FACS series, DakoCytomation Moflo, or similar instrument)
  • Additional reagents and equipment for isolation of PBMC by Ficoll‐Hypaque gradient centrifugation (unit 7.1) and trypan blue exclusion test of cell viability ( appendix 3B)

Alternate Protocol 1: Enrichment of NK Cells

  • Enriched NK cells (not previously stained with an antibody)
  • Staining buffer (see recipe)
  • Anti‐CD56 fluorescently conjugated Ab (NKH1 clone, Coulter, preferably PE‐ or PC5‐conjugated)
  • Fluorescently conjugated isotype control–Ab (mouse IgG, Coulter)
  • 5‐ml flow cytometry tubes
  • Flow cytometer (Coulter, Becton‐Dickinson, DakoCytomation)

Support Protocol 1: Titration of Fluorescently Conjugated Monoclonal Antibodies

  • P815 cell line (ATCC# TIB‐64)
  • Rabbit anti‐mouse lymphocyte Ab (Accurate Chemical)
  • RPMI‐10 ( appendix 2A)
  • 51Chromium51 (Cr)
  • Additional reagents and equipment for analysis of ADCC activity (unit 7.27) and counting cells by trypan blue exclusion ( appendix 3B)
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Literature Cited

Literature Cited
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