Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay

Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the... Background Neutrophil subpopulations with enhanced oxidative reactivity have been described in a number of clinical and in vitro settings. In the dichlorofluorescin (DCFH) oxidation assay, it is essential to maintain cellular viability and plasma membrane integrity through all stages of sample preparation. The process of erythrocyte lysing is crucial because a number of commercial lysing reagents can increase leukocyte membrane permeability. Methods We assessed viability (propidium iodide (PI) method), DCFH oxidation, and CD11b expression of resting or in vitro–stimulated neutrophils exposed to six different red cell lysing procedures. Results Formaldehyde‐containing reagents (Optilyse B, FACS Lyse, and Erythrolyse) but not hypotonic shock or ammonium chloride (NH4Cl) solutions rendered 91.4–99.8% of resting neutrophils PI positive, with concomitant reductions in dichlorofluorescein (DCF) fluorescence, suggesting efflux of the fluorochrome. However, when stimulated with N‐formyl‐methionyl‐leucyl‐phenylalanine or Yersinia enterocolitica and then treated with FACS Lyse or Erythrolyse, up to 69.9% of neutrophils remained PI negative and exhibited enhanced DCF fluorescence. CD11b expression of PI‐positive and ‐negative neutrophils was comparable, suggesting that they were activated equally. Conclusions FACS Lyse and Erythrolyse can modify neutrophil plasma membrane integrity, whereas hypotonic shock and NH4Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay. Cytometry 43:290–296, 2001. © 2001 Wiley‐Liss, Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cytometry Wiley

Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay

Cytometry, Volume 43 (4) – Apr 1, 2001

Loading next page...
 
/lp/wiley/evaluation-of-red-blood-cell-lysing-solutions-in-the-study-of-uBeEQT6Qe0
Publisher
Wiley
Copyright
Copyright © 2001 Wiley‐Liss, Inc.
ISSN
1552-4922
eISSN
1552-4930
DOI
10.1002/1097-0320(20010401)43:4<290::AID-CYTO1061>3.3.CO;2-O
Publisher site
See Article on Publisher Site

Abstract

Background Neutrophil subpopulations with enhanced oxidative reactivity have been described in a number of clinical and in vitro settings. In the dichlorofluorescin (DCFH) oxidation assay, it is essential to maintain cellular viability and plasma membrane integrity through all stages of sample preparation. The process of erythrocyte lysing is crucial because a number of commercial lysing reagents can increase leukocyte membrane permeability. Methods We assessed viability (propidium iodide (PI) method), DCFH oxidation, and CD11b expression of resting or in vitro–stimulated neutrophils exposed to six different red cell lysing procedures. Results Formaldehyde‐containing reagents (Optilyse B, FACS Lyse, and Erythrolyse) but not hypotonic shock or ammonium chloride (NH4Cl) solutions rendered 91.4–99.8% of resting neutrophils PI positive, with concomitant reductions in dichlorofluorescein (DCF) fluorescence, suggesting efflux of the fluorochrome. However, when stimulated with N‐formyl‐methionyl‐leucyl‐phenylalanine or Yersinia enterocolitica and then treated with FACS Lyse or Erythrolyse, up to 69.9% of neutrophils remained PI negative and exhibited enhanced DCF fluorescence. CD11b expression of PI‐positive and ‐negative neutrophils was comparable, suggesting that they were activated equally. Conclusions FACS Lyse and Erythrolyse can modify neutrophil plasma membrane integrity, whereas hypotonic shock and NH4Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay. Cytometry 43:290–296, 2001. © 2001 Wiley‐Liss, Inc.

Journal

CytometryWiley

Published: Apr 1, 2001

Keywords: neutrophils; membrane integrity; free radicals; FACS

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create folders to
organize your research

Export folders, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off