Isolation of Human Basophils

John T. Schroeder1, Anja P. Bieneman1

1 Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.24
DOI:  10.1002/0471142735.im0724s112
Online Posting Date:  February, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Isolating human basophils from blood has long been hampered by the fact that these granulocytes represent just 1% or less of the circulating leukocyte population. We describe herein laboratory protocols that have been refined over the past ∼25 years that now enable investigators to prepare basophils for use in a variety of assays to assess the in vitro biology of these immune cells, both in IgE ‐dependent and ‐independent responses. © 2016 by John Wiley & Sons, Inc.

Keywords: immunologic studies in humans; in vitro assays for immune cell function; innate immunity; immune disease

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Basophil Enrichment by Percoll Gradient Centrifugation
  • Alternate Protocol 1: Basophil Purification Using Negative Selection with Immunomagnetic Beads
  • Support Protocol 1: Percoll Gradient Preparation
  • Support Protocol 2: Alcian Blue and Staining Procedure
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Basophil Enrichment by Percoll Gradient Centrifugation

  Materials
  • Venous blood, freshly drawn, preferably into EDTA‐loaded syringes or purple‐top Vacutainer tubes
  • PAG‐EDTA buffer (see recipe)
  • Percoll gradients in 50–ml polystyrene conical tube (see protocol 3)
  • PAG buffer (see recipe), cold
  • 50‐ml polypropylene conical tubes (e.g., Corning Falcon) pre‐coated with 3 to 5 ml PAG buffer for at least 15 min (the human serum albumin in PAG will coat plastic and prevent cells from sticking)
  • Centrifuge with rotor for swinging bucket carriers and manual slow start‐up feature (e.g., tabletop model HN‐SII from IEC, or equivalent)
  • 3‐ml disposable polyethylene transfer pipets (Fisher Scientific, cat. no. 13‐711‐7M)
  • 15‐ml polystyrene conical tubes pre‐coated with 3 to 5 ml PAG buffer for at least 15 min (the human serum albumin in PAG will coat plastic and prevent cells from sticking)
  • Additional reagents and equipment for Alcian blue staining (see protocol 4) and counting cells using a hemacytometer ( appendix 3A; Strober, )

Alternate Protocol 1: Basophil Purification Using Negative Selection with Immunomagnetic Beads

  Additional Materials (also see protocol 1Basic Protocol)
  • Column buffer (see recipe)
  • Antibody cocktail and bead kit (Miltenyi Biotec, cat. no. 130‐092‐662)
  • LS column (Miltenyi Biotec, cat. no. 130‐042‐401)
  • MidiMACS or QuadraMACS magnetic separator (Miltenyi Biotech)

Support Protocol 1: Percoll Gradient Preparation

  Materials
  • Percoll (GE Healthcare)
  • 10× PIPES (see recipe)
  • 1× PIPES [mix 1 part 10× PIPES (see recipe) with 9 parts deionized, distilled H 2O]
  • 500‐ml container
  • 50–ml polystyrene conical tubes (e.g., AccuSpin tubes from Sigma Chemical, optional)
  • 9‐in. glass Pasteur pipets with small opening at narrow‐end tip: made by heating over the blue flame of a Bunsen burner and pulling on both ends of the pipet; a small opening (∼0.1 mm) is made at the tip by gently clipping with scissors

Support Protocol 2: Alcian Blue and Staining Procedure

  Materials
  • 0.1 M EDTA stock (see recipe)
  • Normal saline: 0.9% (w/v) NaCl
  • Cetyl pyridinium chloride (C 21H 38ClN)
  • Lanthanum chloride (LaCl 3·7H 2O)
  • Alcian Blue (Acros Organics, cat. no. 400460100)
  • Sodium chloride (NaCl)
  • Hydrochloric acid (HCl)
  • Cell suspension to be counted
  • 0.5‐ml microcentrifuge tubes
  • Hemacytometer (Spiers‐Levy recommended)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A; Strober, )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Frischmeyer‐Guerreio, P.A. and Schroeder, J.T. 2012. Cellular immune response parameters that influence IgE sensitization. J. Immunol. Methods 383:21‐29. doi: 10.1016/j.jim.2011.12.007.
  Gilbert, H.S. and Ornstein, L. 1975. Basophil counting with a new staining method using alcian blue. Blood 46:279‐286.
  Hofman, F.M. and Taylor, C.R. 2013. Immunohistochemistry. Curr. Protoc. Immunol. 103:21.4.1‐21.4.26.
  Marone, G., Borriello, F., Varricchi, G., Genovese, A., and Granata, F. 2014. Basophils: Historical reflections and perspective. Chem. Immunol. Allergy 100:172‐192. doi: 10.1159/000358734.
  McGowan, E.C. and Saini, S. 2013. Update on the performance and application of the basophil activation tests. Curr. Allergy Asthma Rep. 13:101‐109. doi: 10.1007/s11882‐012‐0324‐x.
  Schroeder, J.T. and A. Kagey‐Sobotka. 2001. Assay methods for the measurement of mediators and markers of allergic inflammation. In Manual of Clinical Laboratory Immunology, 6th ed. (N.R. Rose, E.C. deMacario, J.D. Folds, H.C. Lane, and R.M. Nakamura eds.). American Society for Microbiology Press, Washington, D.C.
  Strober, W. 1997. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library