Document Detail


Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay.
MedLine Citation:
PMID:  21315723     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.
Authors:
R Keith Reeves; Tristan I Evans; Jacqueline Gillis; Fay E Wong; Michelle Connole; Angela Carville; R Paul Johnson
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2011-02-18
Journal Detail:
Title:  Journal of immunological methods     Volume:  367     ISSN:  1872-7905     ISO Abbreviation:  J. Immunol. Methods     Publication Date:  2011 Mar 
Date Detail:
Created Date:  2011-04-05     Completed Date:  2011-06-02     Revised Date:  2013-06-30    
Medline Journal Info:
Nlm Unique ID:  1305440     Medline TA:  J Immunol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  95-8     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier B.V. All rights reserved.
Affiliation:
Division of Immunology, New England Primate Research Center, Harvard Medical School, Southborough Campus, Pine Hill Drive, Southborough, MA 01772, United States.
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MeSH Terms
Descriptor/Qualifier:
Animals
Antigens, CD45 / analysis
Flow Cytometry / methods*
Fluorescence
HLA-DR Antigens / analysis
Leukocyte Count
Leukocytes, Mononuclear / cytology*
Macaca mulatta
Mucous Membrane / cytology
Reproducibility of Results
Grant Support
ID/Acronym/Agency:
AI071306/AI/NIAID NIH HHS; AI090735/AI/NIAID NIH HHS; P01 AI071306-05/AI/NIAID NIH HHS; P51 RR000168-50/RR/NCRR NIH HHS; R01 AI090735-02/AI/NIAID NIH HHS; RR00168/RR/NCRR NIH HHS; U19 AI 067854-04/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/HLA-DR Antigens; EC 3.1.3.48/Antigens, CD45
Comments/Corrections

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