Isolation of Mouse Neutrophils

Muthulekha Swamydas1, Yi Luo2, Martin E. Dorf2, Michail S. Lionakis1

1 Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.20
DOI:  10.1002/0471142735.im0320s110
Online Posting Date:  August, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation‐based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence‐activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2015 by John Wiley & Sons, Inc.

Keywords: neutrophils; mouse bone marrow; mouse tissue; density gradient centrifugation; magnetic separation; sorting; isolation

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow
  • Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues
  • Alternate Protocol 1: Isolation of Neutrophils Using FACS
  • Basic Protocol 3: Isolation of Neutrophils from Peritoneal Fluid
  • Basic Protocol 4: Isolation of Neutrophils from Peripheral Blood
  • Alternate Protocol 2: Isolation of Neutrophils from Peritoneal Fluid or Peripheral Blood
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow

  Materials
  • C57BL/6 mice (control and Candida albicans‐infected; see unit ; optional)
  • 70% (v/v) ethanol solution
  • 1 × RPMI 1640 with L‐glutamine and 25 mM HEPES (Mediatech, cat. no. 10‐041‐CV)
  • Heat‐inactivated fetal bovine serum (FBS; Gemini Bioproducts, cat. no. 100‐106)
  • Penicillin/streptomycin (10,000 U penicillin/10,000 μg/ml streptomycin) (Mediatech, cat. no. 30‐002‐CI)
  • Ice
  • Phosphate‐buffered saline (PBS) without calcium and magnesium (Mediatech, cat. no. 21‐040‐CV)
  • EDTA (Mediatech, cat. no. 46034‐CI)
  • 0.2% and 1.6% sodium chloride (NaCl) solutions in distilled water (J.T. Baker, cat. no. 3624‐01)
  • Sterile Histopaque 1119 (Sigma‐Aldrich, cat. no. 11191)
  • Sterile Histopaque 1077 (Sigma‐Aldrich, cat. no. 10771)
  • LIVE/DEAD fixable blue dead cell stain kit (Invitrogen, cat. no. L‐23105)
  • Anti‐mouse CD45 APC (clone 30‐F11; BD Biosciences, cat. no. 559864)
  • Anti‐mouse Ly6G PE (clone 1A8; BD Biosciences, cat. no. 551481)
  • Anti‐mouse CD11b APC‐eFluor® 780 (clone M1/70; eBioscience, cat. no. 47‐0112‐82)
  • Tweezers, scalpels, and scissors, kept in 70% ethanol solution
  • 100 × 15–mm petri dishes (BD Falcon, cat. no. 351029)
  • 25‐G × 5/8‐in. needles (BD Biosciences, cat. no. 305122)
  • 12‐ml syringes (Kendall, cat. no. 512878)
  • 50‐ml conical centrifuge tubes (BD Falcon, cat. no. 352098)
  • 100‐μm cell strainers (BD Biosciences, cat. no. 352360)
  • Combitips Plus Biopur (Eppendorf, cat. no. 2249608‐5)
  • Refrigerated and room temperature centrifuges
  • 15‐ml conical centrifuge tubes (Corning, cat. no. 430053)
  • Tissue culture hood
  • 25‐ml serological pipets (BD Falcon, cat. no. 356525)
  • 10‐ml serological pipets (BD Falcon, cat. no. 356551)
  • 3‐ml Pasteur pipets (BD Falcon, cat. no. 357575)
  • Additional reagents and equipment for euthanasia of mice (unit ), infection of mice with Candida albicans (unit ), and counting viable cells by trypan blue exclusion ( )
NOTE: All prepared solutions can be sterile‐filtered using 0.2‐μm filters and stored at 4°C.

Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues

  Materials
  • C57BL/6 mice
  • 10 × phosphate‐buffered saline (PBS; Mediatech, cat. no. 46‐013‐CM)
  • 1 × RPMI 1640 with L‐glutamine and 25 mM HEPES (Mediatech, cat. no. 10‐041‐CV)
  • Heat‐inactivated fetal bovine serum (FBS; Gemini Bioproducts, cat. no. 100‐106)
  • Penicillin/streptomycin (10,000 U penicillin/10,000 μg/ml streptomycin) (Mediatech, cat. no. 30‐002‐CI)
  • Ice
  • RPMI without serum
  • Liberase TL (Roche, cat. no. 05401020001)
  • DNase I (Roche, cat. no. 10104159001)
  • FACS buffer: Phosphate‐buffered saline (PBS) without calcium and magnesium supplemented with 0.5% bovine serum albumin (BSA) and 0.01% sodium azide [10% (w/v) sodium azide (Teknova, cat. no. S0209)]
  • Percoll (GE Healthcare, cat. no. 17‐0891‐01)
  • Magnetic‐activated cell sorting (MACS) buffer: PBS containing 2 mM EDTA (Mediatech, cat. no. 46034‐CI) and 0.5% bovine serum albumin (BSA)
  • Anti‐Ly6G microbead kit for mouse neutrophils (Miltenyi, cat. no. 130‐092‐332)
  • LIVE/DEAD fixable blue dead cell stain kit (Invitrogen, cat. no. L‐23105)
  • Anti‐mouse CD45 APC (clone 30‐F11; BD Biosciences, cat. no. 559864)
  • Anti‐mouse Ly6G PE (clone 1A8; BD Biosciences, cat. no. 551481)
  • Anti‐mouse CD11b APC‐eFluor® 780 (clone M1/70; eBioscience, cat. no. 47‐0112‐82)
  • Anti‐mouse CD16/CD32 (clone 93; eBioscience, cat. no. 14‐0641‐86)
  • Surgical instruments (kept in 70% ethanol solution) including:
    • Tweezers
    • Scalpels
    • Scissors
  • 100 × 15–mm petri dishes (BD Falcon, cat. no. 351029)
  • 50‐ml conical centrifuge tubes (BD Falcon, cat. no. 352098)
  • 37°C water bath
  • 40‐μm cell strainers (BD Biosciences, cat. no. 352340)
  • 70‐μm cell strainers (BD Biosciences, cat. no. 352350)
  • 100‐μm cell strainers (BD Biosciences, cat. no. 352360)
  • Refrigerated and room temperature centrifuges
  • 15‐ml conical centrifuge tubes (BD Falcon, cat. no. 352097)
  • LS+ positive selection columns (Miltenyi, cat. no. 130‐042‐401)
  • QuadroMACS Separator (Miltenyi, cat. no. 130‐090‐976)
  • MACS MultiStand (Miltenyi cat # 130‐042‐303)
  • Tissue culture hood
  • 25‐ml serological pipets (BD Falcon, cat. no. 356525)
  • 10‐ml serological pipets (BD Falcon, cat. no. 356551)
  • 3‐ml Pasteur pipets (BD Falcon, cat. no. 357575)
  • 12‐ml syringes (Kendall, cat. no. 512878)
  • 25‐G × 5/8‐in. needles (BD Biosciences, cat. no. 305122)
  • Additional reagents and equipment for euthanasia of mice (unit ) and counting viable cells by trypan blue exclusion ( ).
NOTE: All prepared solutions can be stored up to 3 months at 4°C. Please follow manufacturer's instructions for storage of antibodies and media purchased.NOTE: Prepare single cell suspensions of mouse kidney, live or spleen.

Alternate Protocol 1: Isolation of Neutrophils Using FACS

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • Anti‐mouse CD45 APC (clone 30‐F11; BD Biosciences, cat. no. 559864)
  • Anti‐mouse Ly6G PE (clone 1A8; BD Biosciences, cat. no. 551481)
  • Anti‐mouse CD11b APC‐eFluor® 780 (clone M1/70; eBioscience, cat. no. 47‐0112‐82)
  • Anti‐mouse CD16/CD32 (clone 93; eBioscience, cat. no. 14‐0641‐86)
  • Sorting buffer: PBS without calcium and magnesium supplemented with 10% FBS and 0.5 mM EDTA
  • FACS tubes (BD Biosciences, cat. no. 352053)
  • Additional reagents and equipment for flow cytometry and FACS (Chapter 5)

Basic Protocol 3: Isolation of Neutrophils from Peritoneal Fluid

  Materials
  • Casein solution (see recipe)
  • Donor mice (2‐ to 4‐month‐old male mice of any conventional strain)
  • Harvest solution: 0.02% EDTA in 1 × PBS (see recipe for 10 ×), filter‐sterilized through 0.2‐μm filter (room temperature)
  • 70% ethanol
  • 1 × PBS (see recipe for 10 ×)
  • Percoll gradient solution (see recipe), room temperature
  • Medium to be used with neutrophils in subsequent experimental procedures, room temperature
  • Medium to be used with neutrophils in subsequent experimental procedures, containing 50% (v/v) FBS, room temperature
  • Diff‐Quik staining solutions (Baxter)
  • 10‐ml syringes and 21‐G needles
  • Forceps
  • Small, straight surgical scissors
  • 15‐ or 50‐ml conical polypropylene centrifuge tubes
  • Benchtop centrifuge
  • 10‐ml ultracentrifuge tubes (Beckman or equivalent)
  • Sorvall RC70 ultracentrifuge with T‐1270 fixed‐angle rotor (or equivalent)
  • Cytospin cytocentrifuge including chambers and filter cards (Shandon/Lipshaw)
  • Microscope slides appropriate for use with Cytospin centrifuge
  • Additional reagents and equipment for intraperitoneal injection (unit ), euthanasia of mice (unit ), harvesting peritoneal exudate cells (unit ), and counting viable cells by trypan blue exclusion ( )

Basic Protocol 4: Isolation of Neutrophils from Peripheral Blood

  Materials
  • Naive mice
  • Methoxyflurane (Metofane)
  • Heparin
  • 1 × phosphate‐buffered saline (PBS; see recipe for 10 ×)
  • 1‐ml syringe with 20‐ to 22‐G needle
  • 12 × 75‐mm glass tube
  • Benchtop centrifuge
  • Additional reagents and equipment for methoxyflurane anesthesia of mice (unit ), cardiac puncture of mice (unit ), euthanasia of mice (unit ), counting cells using a hemacytometer ( ), and isolation of neutrophils by density gradient centrifugation (as for peritoneal fluid; see protocol 4, steps 9 to 14)

Alternate Protocol 2: Isolation of Neutrophils from Peritoneal Fluid or Peripheral Blood

  Additional Materials (also see Basic Protocols protocol 43 and protocol 54)
  • Histopaque‐1119 and ‐1077 (polysucrose/sodium diatrizoate, densities 1.1119 and 1.077, respectively; store at 4°C; Sigma)
  • 15‐ml polypropylene centrifuge tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Amulic, B. , Cazalet, C. , Hayes, G.L. , Metzler, K.D. , and Zychlinsky, A. 2012. Neutrophil function: From mechanisms to disease. Annu. Rev. Immunol. 30:459‐489.
   Boxio, R. , Bossenmeyer‐Pourie, C. , Steinckwich, N. , Dournon, C. , and Nusse, O. 2004. Mouse bone marrow contains large numbers of functionally competent neutrophils. J. Leukoc. Biol. 75:604‐611.
   Clark, R.A. and Nauseef, W.M. 2001. Isolation and functional analysis of neutrophils. Curr. Protoc. Immunol. 19:7.23.1‐7.23.17.
   Daley, J.M. , Thomay, A.A. , Connolly, M.D. , Reichner, J.S. , and Albina, J.E. 2008. Use of Ly6G‐specific monoclonal antibody to deplete neutrophils in mice. J. Leukoc. Biol. 83:64‐70.
   Devi, S. , Laning, J. , Luo, Y. , and Dorf, M.E. 1995. Biologic activities of the β‐chemokine TCA3 on neutrophils and macrophages. J. Immunol. 154:5376‐5383.
   Kruger, P. , Saffarzadeh, M. , Weber, A.N. , Rieber, N. , Radsak, M. , von Bernuth, H. , Benarafa, C. , Roos, D. , Skokowa, J. , and Hartl, D. 2015. Neutrophils: Between host defence, immune modulation, and tissue injury. PLoS Pathog. 11:e1004651.
   Lionakis, M.S. , Lim, J.K. , Lee, C.C. , and Murphy, P.M. 2011. Organ‐specific innate immune responses in a mouse model of invasive candidiasis. J. Innate Immun. 3:180‐199.
   Lionakis, M.S. , Fischer, B.G. , Lim, J.K. , Swamydas, M. , Wan, W. , Richard Lee, C.C. , Cohen, J.I. , Scheinberg, P. , Gao, J.L. , and Murphy, P.M. 2012. Chemokine receptor Ccr1 drives neutrophil‐mediated kidney immunopathology and mortality in invasive candidiasis. PLoS Pathog. 8:e1002865.
   Luo, Y. and Dorf, M.E. 2001. Isolation of mouse neutrophils. Curr. Protoc. Immunol. 22:3.20.1‐3.20.6.
   Narasaraju, T. , Yang, E. , Samy, R.P. , Ng, H.H. , Poh, W.P. , Liew, A.A. , Phoon, M.C. , van Rooijen, N. , and Chow, V.T. 2011. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis. Am. J. Pathol. 179:199‐210.
   Watt, S.M. , Burgess, A.W. , and Metcalf, D. 1979. Isolation and surface labeling of murine polymorphonuclear neutrophils. J. Cell Physiol. 100:1‐21.
   Whitney, P.G. , Bar, E. , Osorio, F. , Rogers, N.C. , Schraml, B.U. , Deddouche, S. , LeibundGut‐Landmann, S. , and Reis e Sousa, C. 2014. Syk signaling in dendritic cells orchestrates innate resistance to systemic fungal infection. PLoS Pathog. 10:e1004276.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library