Wright‐Giemsa and Nonspecific Esterase Staining of Cells

Warren Strober1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Appendix 3D
DOI:  10.1002/0471142956.cya03ds11
Online Posting Date:  May, 2001
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Abstract

This appendix provides two protocols for staining cells. The stains used in the Wright‐Giemsa protocol, the Romanowsky stains, are a mixture of methylene blue (and other closely related thiazine dyes) and eosin. The staining protocol is a two‐stage method that allows for a more intense staining of the nuclei than would be possible if the Wright and Giemsa stains were mixed together. In the second protocol, nonspecific esterase stain are used to identify cell types containing esterases that have a characteris termediate that can be coupled with a substance, hexazotized pararosaniline, to yield a colored precipitate. Thus, this staining reaction can be used to identify monocytes/macrophages in cell preparations.

     
 
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Table of Contents

  • Basic Protocol 1: Cytospin Preparation
  • Basic Protocol 2: Wright‐Giemsa Staining
  • Basic Protocol 3: Nonspecific Esterase Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Cytospin Preparation

  Materials
  • 5 × 106 cell/ml suspension of cells of interest
  • 22% (w/v) bovine serum albumin (BSA)
  • Cytocentrifuge (e.g., Cytospin 3; Shandon/Lipshaw) and standard microscope slides

Basic Protocol 2: Wright‐Giemsa Staining

  Materials
  • 100% methanol
  • Wright stain (Columbia Diagnostics)
  • Working buffer (see recipe)
  • Working Giemsa stain (see recipe)
CAUTION: Methanol is toxic and can cause blindness if swallowed.

Basic Protocol 3: Nonspecific Esterase Staining

  Materials
  • Buffered formaldehyde‐acetone fixative (see recipe), ice‐cold
  • Phosphate buffer/hexazotized pararosaniline/α‐naphthyl butyrate (see recipe)
  • Gill's hematoxylin (Columbia Diagnostics)
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Figures

Videos

Literature Cited

Literature Cited
   Li, C.Y., Lam, K.W., and Yam, L.T. 1973. Esterases in human leukocytes. J. Histochem. Cytochem. 21:1‐12.
   Marshall, P.N., Bentley, S.A., and Lewis, S.M. 1975. An evaluation of some commercial Romanowsky stains. J. Clin. Pathol. (Lond.) 28:680.
   Wittekind, D. 1979. On the nature of Romanowsky dyes and the Romanowsky‐Giemsa effect. Clin. Lab. Haemat. 1:247‐262.
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