Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry

Melissa L. Maes, Lisa B. Davidson, Paul F. McDonagh, Leslie S Ritter

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 μg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5 μg/ml PerCP-conjugated anti-CD45, 5 μg/ml FITC-conjugated anti-CD11b, or 80 μM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior to antibody staining caused a decrease in overall fluorescence; it can be used to successfully identify extra-cellular markers.

Original languageEnglish (US)
Pages (from-to)79-86
Number of pages8
JournalJournal of Immunological Methods
Volume319
Issue number1-2
DOIs
StatePublished - Jan 30 2007

Fingerprint

Dye Lasers
Flow Cytometry
Leukocytes
Fluorescence
Staining and Labeling
Formaldehyde
Phosphates
Monoclonal Antibodies
CD45 Antigens
Fc Receptors
Fluorescein-5-isothiocyanate
Granulocytes
Nucleic Acids
Permeability
Blood Cells
Membrane Proteins
Coloring Agents
Erythrocytes
Antibodies

Keywords

  • CD11b
  • Flow cytometry
  • LDS-751
  • Mouse
  • Neutrophil
  • Paraformaldehyde
  • Reactive oxidative species

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry. / Maes, Melissa L.; Davidson, Lisa B.; McDonagh, Paul F.; Ritter, Leslie S.

In: Journal of Immunological Methods, Vol. 319, No. 1-2, 30.01.2007, p. 79-86.

Research output: Contribution to journalArticle

@article{7f2f013231e9424babd5a8920508b8ae,
title = "Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry",
abstract = "Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 μg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5 μg/ml PerCP-conjugated anti-CD45, 5 μg/ml FITC-conjugated anti-CD11b, or 80 μM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior to antibody staining caused a decrease in overall fluorescence; it can be used to successfully identify extra-cellular markers.",
keywords = "CD11b, Flow cytometry, LDS-751, Mouse, Neutrophil, Paraformaldehyde, Reactive oxidative species",
author = "Maes, {Melissa L.} and Davidson, {Lisa B.} and McDonagh, {Paul F.} and Ritter, {Leslie S}",
year = "2007",
month = "1",
day = "30",
doi = "10.1016/j.jim.2006.10.016",
language = "English (US)",
volume = "319",
pages = "79--86",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry

AU - Maes, Melissa L.

AU - Davidson, Lisa B.

AU - McDonagh, Paul F.

AU - Ritter, Leslie S

PY - 2007/1/30

Y1 - 2007/1/30

N2 - Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 μg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5 μg/ml PerCP-conjugated anti-CD45, 5 μg/ml FITC-conjugated anti-CD11b, or 80 μM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior to antibody staining caused a decrease in overall fluorescence; it can be used to successfully identify extra-cellular markers.

AB - Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 μg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5 μg/ml PerCP-conjugated anti-CD45, 5 μg/ml FITC-conjugated anti-CD11b, or 80 μM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior to antibody staining caused a decrease in overall fluorescence; it can be used to successfully identify extra-cellular markers.

KW - CD11b

KW - Flow cytometry

KW - LDS-751

KW - Mouse

KW - Neutrophil

KW - Paraformaldehyde

KW - Reactive oxidative species

UR - http://www.scopus.com/inward/record.url?scp=33846240409&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846240409&partnerID=8YFLogxK

U2 - 10.1016/j.jim.2006.10.016

DO - 10.1016/j.jim.2006.10.016

M3 - Article

C2 - 17187818

AN - SCOPUS:33846240409

VL - 319

SP - 79

EP - 86

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 1-2

ER -