Isolation of Human Eosinophils

Praveen Akuthota1, Revital Shamri1, Peter F. Weller1

1 Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.31
DOI:  10.1002/0471142735.im0731s98
Online Posting Date:  August, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Highly purified eosinophils can be isolated from peripheral blood by negative selection using an antibody‐based magnetic negative selection protocol. The basic protocol describes a sequential fractionation of peripheral blood in which CD16+ granulocytes are enriched first from whole blood, followed by isolation of eosinophils. This technique is easy to use, fast, and highly reproducible. Support protocols describe a staining methods that can be used to evaluate the purity of eosinophils and differentiation from other leukocyte populations. Curr. Protoc. Immunol. 98:7.31.1‐7.31.8. © 2012 by John Wiley & Sons, Inc.

Keywords: eosinophils; cell separation; staining; labeling

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Antibody‐Based Magnetic Negative Selection of Eosinophils
  • Support Protocol 1: Fast Green and Neutral Red Staining of Eosinophils
  • Support Protocol 2: Hema 3 Staining of Eosinophils
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Antibody‐Based Magnetic Negative Selection of Eosinophils

  Materials
  • Human blood donor
  • Acidified sodium citrate (see recipe), sterile
  • 6% Dextran 70 in 0.9% NaCl (Pharmacosmos)
  • Ficoll‐Paque Premium (GE Healthcare)
  • HBSS without Ca2+, Mg2+, or phenol red ( appendix 2A), 4°C
  • 0.2% NaCl and 1.6% NaCl, 4°C
  • Turk Blood Diluting Fluid (Ricca Chemical Company)
  • Human eosinophil enrichment kit (StemCell Technologies; negative selection kit with negative selection antibody cocktail) including:
    • Monoclonal antibodies in cocktail include: anti‐CD2 (to deplete residual T cells), anti‐CD14 (to deplete residual monocytes), anti‐CD16 (to deplete residual neutrophils), anti‐CD19 (to deplete residual B‐cells), anti‐CD56 (to deplete residual NK cells), anti‐glycophorin A (to deplete residual red blood cells), and anti‐dextran
    • Magnetic colloid
  • Separation medium: 0.5% (w/v) OVA in HBSS without Ca2+, Mg2+, or phenol red ( appendix 2A), 4°C (alternatively, PBS with 2% FBS is available from StemCell Technologies as a separation medium and may be used)
  • Sterile 60‐ml Luer‐lock syringes
  • 19‐G butterfly needles
  • 50‐ml conical polypropylene centrifuge tubes
  • Benchtop refrigerated centrifuge for 50‐ml conical tubes (Eppendorf 5810R or equivalent)
  • Sterile disposable transfer pipets
  • Magnetic separation apparatus capable of accommodating 0.5‐ or 0.6‐in. magnetic columns (StemCell Technologies) kept in cold room
  • 0.5‐ or 0.6‐in. negative selection gravity‐feed column (StemCell Technologies) with 3‐way Luer stopcock assembly
  • 21‐G needles
  • Additional reagents and equipment for obtaining peripheral blood by venipuncture ( appendix 3F), counting cells with a hemacytometer ( appendix 3A), and eosinophil staining with fast green/neutral red (or Hema 3 eosin‐based staining, see Support Protocols protocol 21 and protocol 32)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.

Support Protocol 1: Fast Green and Neutral Red Staining of Eosinophils

  Materials
  • Purified eosinophil suspension (see protocol 1)
  • Methanol
  • 0.2% (w/v) fast green (Sigma‐Aldrich) in 70% ethanol (store up to 1 month at room temperature in polypropylene tube)
  • 0.5% (w/v) neutral red (Sigma‐Aldrich) in distilled water (store up to 1 month at room temperature in polypropylene tube)
  • Microscope slides
  • Cytocentrifuge (Cytospin, Shandon/Lipshaw)

Support Protocol 2: Hema 3 Staining of Eosinophils

  Materials
  • Purified eosinophil suspension (see protocol 1)
  • Deionized water
  • Hema 3 kit (Fisher Scientific, cat. no.122‐911) containing:
    • Fixative solution: 0.0002% (w/v) fast green in methanol
    • Solution 1: contains 0.125% (w/v) eosin Y, <0.55% (w/v) potassium phosphate monobasic, <0.4% (w/v) sodium phosphate dibasic, 0.01% sodium azide, in water
    • Solution 2: contains 0.0625% (w/v) azure A, 0.0625% (w/v) methylene blue, <0.55% (w/v) potassium phosphate monobasic, <0.4% (w/v) sodium phosphate dibasic, 0.01% sodium azide, in water
  • Microscope slides
  • Cytocentrifuge (Cytospin, Shandon/Lipshaw)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Blom, M., Tool, A., Mul, F., Knol, E., Roos, D., and Verhoeven, A. 1995. Eosinophils isolated with two different methods show different characteristics of activation. J. Immunol. Methods 178:183‐193.
   Day, R. 1970. Eosinophil cell separation from human peripheral blood. Immunology 18:955.
   Floyd, H., Ni, J., Cornish, A.L., Zeng, Z., Liu, D., Carter, K.C., Steel, J., and Crocker, P.R. 2000. Siglec‐8. A novel eosinophil‐specific member of the immunoglobulin superfamily. J. Biol. Chem. 275:861‐866.
   Gartner, I. 1980. Separation of human eosinophils in density gradients of polyvinylpyrolidone‐coated silica gel (Percoll). Immunology 40:133‐136.
   Hansel, T., Pound, J., and Thompson, R. 1990. Isolation of eosinophils from human blood. J. Immunol. Methods 127:153‐164.
   Hansel, T.T., de Vries, I.J.M., Iff, T., Rihs, S., Wandzilak, M., Betz, S., Blaser, K., and Walker, C. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145:105‐110.
   Hartnell, A., Kay, A.B., and Wardlaw, A.J. 1992. IFN‐gamma induces expression of Fc gamma RIII (CD16) on human eosinophils. J. Immunol. 148:1471‐1478.
   Ide, M., Weiler, D., Kita, H., and Gleich, G.J. 1994. Ammonium chloride exposure inhibits cytokine‐mediated eosinophil survival. J. Immunol. Methods 168:187‐196.
   Jackson, M.H., Millar, A.M., Dawes, J., and Bell, D. 1989. Neutrophil activation during cell separation procedures. Nucl. Med. Commun. 10:901‐904.
   Kitaura, M., Nakajima, T., Imai, T., Harada, S., Combadiere, C., Tiffany, H.L., Murphy, P.M., and Yoshie, O. 1996. Molecular cloning of human eotaxin, an eosinophil‐selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J. Biol. Chem. 271:7725‐7730.
   Lansdorp, P.M. and Thomas, T.E. 1990. Purification and analysis of bispecific tetrameric antibody complexes. Mol. Immunol. 27:659‐666.
   Laviolette, M., Bossé, M., Rocheleau, H., Lavigne, S., and Ferland, C. 1993. Comparison of two modified techniques for purifying blood eosinophils. J. Immunol. Methods 165:253‐261.
   Malm‐Erjefalt, M., Stevens, T.R., Persson, C.G., and Erjefalt, J.S. 2004. Discontinuous Percoll gradient centrifugation combined with immunomagnetic separation obviates the need for erythrocyte lysis and yields isolated eosinophils with minimal granule abnormalities. J. Immunol. Methods. 288:99‐109.
   Miltenyi, S., Müller, W., Weichel, W., and Radbruch, A. 1990. High gradient magnetic cell separation with MACS. Cytometry 11:231‐238.
   Munoz, N.M. and Leff, A.R. 2006. Highly purified selective isolation of eosinophils from human peripheral blood by negative immunomagnetic selection. Nat. Protoc. 1:2613‐2620.
   Sedgwick, J.B., Shikama, Y., Nagata, M., Brener, K., and Busse, W.W. 1996. Effect of isolation protocol on eosinophil function: Percoll gradients versus immunomagnetic beads. J. Immunol. Methods. 198:15‐24.
   Vadas, M.A., David, J.R., Butterworth, A., Pisani, N.T., and Siongkok, T.A. 1979. A new method for the purification of human eosinophils and neutrophils and a comparison of these cells to damage schistosomula of Schistosoma mansoni. J. Immunol. 122:2950‐2958.
   Wang, H.B., Ghiran, I., Matthaei, K., and Weller, P.F. 2007. Airway eosinophils: Allergic inflammation recruited professional antigen‐presenting cells. J. Immunol. 179:7585‐7592.
   Yazdanbakhsh, M., Eckmann, C., Koenderman, L., Verhoeven, A., and Roos, D. 1987. Eosinophils do respond to fMLP. Blood 70:379‐383.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library