Isolation and Functional Analysis of Human Neutrophils

Douglas B. Kuhns1, Debra A. Long Priel1, Jessica Chu2, Kol A. Zarember2

1 Neutrophil Monitoring Lab, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 2 Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.23
DOI:  10.1002/0471142735.im0723s111
Online Posting Date:  November, 2015
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Abstract

This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll‐Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described. © 2015 by John Wiley & Sons, Inc.

Keywords: neutrophil; leukocyte; NOX2; phagocyte; innate immunity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Neutrophils Using Percoll‐Based Density Gradient
  • Alternate Protocol 1: Ficoll‐Paque Premium as a Density Gradient for Isolation of PMN
  • Basic Protocol 2: Analysis of PMN ROS Generation by Luminol‐Enhanced Chemiluminescence
  • Alternate Protocol 2: Quantitative Analysis of O 2 • ¯ Generation Using Superoxide Dismutase‐Inhibitable Ferricytochrome c Reduction
  • Alternate Protocol 3: Analysis of PMN H2O2 Production by Flow Cytometry of Dihydrorhodamine 123 Staining
  • Basic Protocol 3: Assessment of Internalization of Microbes by PMNS
  • Basic Protocol 4: Assessment of Killing of Microbes by PMN
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Neutrophils Using Percoll‐Based Density Gradient

  Materials
  • Anticoagulated human peripheral blood in Vacutainer tubes
  • Hanks’ Balanced Salt Solution without Ca, Mg, or phenol red [HBSS(–); e.g., Life Technologies, cat. no. 14175]
  • 3% dextran (see recipe)
  • 33 mM NaCl
  • 267 mM NaCl
  • 90% Percoll (see recipe)
  • 3% (v/v) acetic acid
  • Differential stain (e.g., Diff‐Quik or Hemacolor)
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes (e.g., Corning Falcon)
  • Centrifuge
  • Automated cell counter or hemacytometer ( ; Strober, )
  • Cytocentrifuge: e.g., Cytospin 3 (Shandon)
  • Additional reagents and equipment for counting cells ( ; Strober, )

Alternate Protocol 1: Ficoll‐Paque Premium as a Density Gradient for Isolation of PMN

  Materials
  • Blood donor
  • Hanks’ Balanced Salt Solution without Ca, Mg, or phenol red [HBSS(–); Lonza, cat. no. 10‐547]
  • Ficoll‐Paque Premium (GE Healthcare)
  • 3% dextran (see recipe)
  • Hypotonic lysis buffer: 13.6 ml 10× PBS(–) (see below) in 600 ml water
  • Re‐equilibration buffer: 108.1 ml 10× PBS (see below) in 500 ml H 2O
  • 10× phosphate‐buffered saline without Ca or Mg [PBS(–); Lonza, cat. no. 17‐515F, or see recipe]
  • Sodium heparin or acid citrate dextrose (ACD‐A) blood collection tubes
  • 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Centrifuge

Basic Protocol 2: Analysis of PMN ROS Generation by Luminol‐Enhanced Chemiluminescence

  Materials
  • PMN ( protocol 1 or protocol 2)
  • RPMI 1640 medium with 2 mM glutamine, without phenol red, supplemented with 10 to 25 mM HEPES, pH 7.4 (e.g., Life Technologies)
  • 55 mM luminol stock solution (see recipe)
  • Stimulus (see annotations to the steps below for examples)
  • 96‐well flat‐bottom white polypropylene plate (e.g., Whatman Uniplate)
  • Luminometer (e.g., Molecular Devices Filtermax F5)

Alternate Protocol 2: Quantitative Analysis of O 2 • ¯ Generation Using Superoxide Dismutase‐Inhibitable Ferricytochrome c Reduction

  Materials
  • 10 mg/ml catalase (see recipe)
  • 200 μM cytochrome c (see recipe)
  • Hanks’ Balanced Salt Solution with Ca and Mg, without phenol red [HBSS(+); Lonza, cat. no. 10‐527]
  • End‐over‐end rotator
  • 1‐ml polypropylene microcentrifuge tubes
  • Refrigerated microcentrifuge

Alternate Protocol 3: Analysis of PMN H2O2 Production by Flow Cytometry of Dihydrorhodamine 123 Staining

  Materials
  • Human blood donor
  • Blood collection tubes with K 2EDTA anticoagulant
  • Lysis buffer (see recipe)
  • Flow buffer (see recipe)
  • 25 mM dihydrorhodamine 123 (see recipe)
  • 10 mg/ml catalase (see recipe)
  • 15‐ml conical polypropylene centrifuge tubes
  • 5‐ml round bottom polypropylene tubes (e.g., Corning Falcon, cat. no. 352063)
  • Centrifuge
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Basic Protocol 3: Assessment of Internalization of Microbes by PMNS

  Materials
  • 70% ethanol
  • Fresh autologous serum (see recipe)
  • Hanks’ Balanced Salt Solution without Ca, Mg, or phenol red [HBSS(–); e.g., Life Technologies, cat. no. 14175]
  • RPMI 1640 medium with 2 mM glutamine, without phenol red, supplemented with 10 to 25 mM HEPES, pH 7.4 (e.g., Life Technologies)
  • Unlabeled or pHrodo‐labeled bacteria (labeling performed per manufacturer's instructions)
  • Phosphate‐buffered saline without Ca or Mg [PBS(−); Lonza, cat. no. 17‐515F, or see recipe]
  • HARLECO Hemacolor Stain Set (methanol fixative, eosin stain, methylene blue stain)
  • Hard set mounting medium (Vectashield or ProLong Antifade)
  • 4% (w/v) paraformaldehyde
  • 1 μg/ml 4',6‐diamidino‐2‐phenylindole (DAPI)
  • Light microscope
  • Round cover glasses (12 mm diameter, No. 1 thickness)
  • 24‐well sterile tissue culture plates
  • Refrigerated centrifuge that holds tissue culture plates
  • Forceps
  • Microscope slides, ethanol‐cleaned
  • Epifluorescence or confocal microscope

Basic Protocol 4: Assessment of Killing of Microbes by PMN

  Materials
  • Staphylococcus aureus 502 A (ATCC; see recipe for preparation of single‐use aliquots)
  • Trypticase soy broth (see recipe)
  • Hanks’ Balanced Salt Solution without Ca, Mg, or phenol red [HBSS(–); Lonza, cat. no. 10‐547]
  • Hanks’ Balanced Salt Solution with Ca and Mg, without phenol red [HBSS(+); Lonza, cat. no. 10‐527]
  • Trypticase soy agar (see recipe)
  • Sterile distilled H 2O, pH 11 (adjusted with 1 M NaOH)
  • Human AB serum (see recipe)
  • PMN from normal subject and patients ( protocol 1 or Alternate Protocol)
  • 1‐ml (P‐1000) aerosol‐barrier pipet tips
  • 100‐ml Erlenmeyer flask with lid
  • Spectrophotometer for OD 650 measurements
  • 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • 10‐ml bottletop dispenser (e.g., Dispensette III; Sigma‐Aldrich)
  • 75 × 100–mm disposable glass tubes with metal caps
  • 60 × 15–mm tissue culture dishes
  • 2‐ml conical microcentrifuge tubes (e.g., Sarstedt, cat. no. 72.694996)
  • End‐over‐end rotator
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Figures

Videos

Literature Cited

Literature Cited
   Haslett, C. , Guthrie, L.A. , Kopaniak, M.M. , Johnston, R.B., Jr. , and Henson, P.M. 1985. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am. J. Pathol. 119:101‐110.
   Nathan, C. 2006. Neutrophils and immunity: Challenges and opportunities. Nat. Rev. Immunol. 6:173‐182. doi: 10.1038/nri1785.
   Nauseef, W.M. 2007. How human neutrophils kill and degrade microbes: An integrated view. Immunol. Rev. 219:88‐102. doi: 10.1111/j.1600‐065X.2007.00550.x
   Parker, L.C. , Prince, L.R. , Buttle, D.J. , and Sabroe, I. 2009. The generation of highly purified primary human neutrophils and assessment of apoptosis in response to Toll‐like receptor ligands. Methods Mol. Biol. 517:191‐204. doi: 10.1007/978‐1‐59745‐541‐1_12.
   Quinn, M.T. and Deleo, F. 2007. Neutrophil Methods and Protocols, 2nd ed. Humana Press, Totowa, N.J.
   Sabroe, I. , Jones, E.C. , Usher, L.R. , Whyte, M.K. , and Dower, S.K. 2002. Toll‐like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: A critical role for monocytes in leukocyte lipopolysaccharide responses. J. Immunol. 168:4701‐4710. doi: 10.4049/jimmunol.168.9.4701
   Sabroe, I. , Prince, L.R. , Jones, E.C. , Horsburgh, M.J. , Foster, S.J. , Vogel, S.N. , Dower, S.K. , and Whyte, M.K. 2003. Selective roles for Toll‐like receptor (TLR)2 and TLR4 in the regulation of neutrophil activation and life span. J. Immunol. 170:5268‐5275. doi: 10.4049/jimmunol.170.10.5268.
   Segal, A.W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol. 23:197‐223. doi: 10.1146/annurev.immunol.23.021704.115653
   Strober, W. 1997. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2.
   Zarember, K.A. and Kuhns, D.B. 2011. Editorial: Will the real neutrophil please stand up? J. Leukoc. Biol. 90:1039‐1041. doi: 10.1189/jlb.0711334.
Key References
   Nathan , 2006. See above.
  For more information on neutrophil biology, these excellent reviews by Nathan, Nauseef, and Segal are highly recommended.
   Nauseef , 2007. See above.
   Segal , 2005. See above.
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