A six-color flow cytometric assay for the analysis of peripheral blood dendritic cells

Stefania Giannelli, Adriano Taddeo, Pietro Presicce, Maria Luisa Villa, Silvia Della Bella

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Background: Flow cytometric analysis of peripheral blood dendritic cells (PBDCs) and their myeloid (mDCs) and plasmacytoid (pDCs) subsets is a less invasive procedure that is acquiring growing clinical relevance. Because dendritic cells (DCs) lack unique lineage markers, current methods that are based on 3- or 4-color assays do not allow multiparametric analysis of DC subsets. In this study a dedicated 6-color assay was developed. Methods: mDCs and pDCs were counted and characterized for the expression of activation/maturation markers by using a single-platform 6-color assay. Whole-blood samples from 20 healthy controls were directly stained with either CD80-FITC/CD40-PE/lineage-PerCP-Cy5.5/CD123-PE-Cy7/ CD11c-APC/HLA-DRAPC-Cy7 or CD86-FITC/CD83-PE/lineage-PerCP-Cy5.5/ CD123-PE-Cy7/CD11c-APC/HLA-DR-APC-Cy7 combination, in the presence of commercial fluorospheres. A dual-platform 3-color assay currently in use was run in parallel for comparison. Results: The 6-color assay provided mDCs and pDCs counts similar to counts obtained by the 3-color assay. Only the 6-color assay could show differential expression of activation markers by mDCs and pDCs, with pDCs expressing lower levels of costimulatory molecules and HLA-DR, but higher levels of CD83. Conclusions: The 6-color assay described here may be a sensitive tool for assessing possible variations in the number and features of mDCs and pDCs whose reciprocal balance is critical in understanding the more detailed orchestration of immune responses.

Original languageEnglish
Pages (from-to)349-355
Number of pages7
JournalCytometry Part B - Clinical Cytometry
Volume74
Issue number6
DOIs
Publication statusPublished - Nov 2008

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Dendritic Cells
Blood Cells
Color
Fluorescein-5-isothiocyanate
HLA-DR Antigens

Keywords

  • Costi-mulatory molecules
  • Multiparametric flow cytometry
  • Myeloid dendritic cells
  • Peripheral blood dendritic cells
  • Plasmacytoid dendritic cells

ASJC Scopus subject areas

  • Cell Biology
  • Histology
  • Pathology and Forensic Medicine
  • Medicine(all)

Cite this

A six-color flow cytometric assay for the analysis of peripheral blood dendritic cells. / Giannelli, Stefania; Taddeo, Adriano; Presicce, Pietro; Villa, Maria Luisa; Della Bella, Silvia.

In: Cytometry Part B - Clinical Cytometry, Vol. 74, No. 6, 11.2008, p. 349-355.

Research output: Contribution to journalArticle

Giannelli, Stefania ; Taddeo, Adriano ; Presicce, Pietro ; Villa, Maria Luisa ; Della Bella, Silvia. / A six-color flow cytometric assay for the analysis of peripheral blood dendritic cells. In: Cytometry Part B - Clinical Cytometry. 2008 ; Vol. 74, No. 6. pp. 349-355.
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AU - Giannelli, Stefania

AU - Taddeo, Adriano

AU - Presicce, Pietro

AU - Villa, Maria Luisa

AU - Della Bella, Silvia

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N2 - Background: Flow cytometric analysis of peripheral blood dendritic cells (PBDCs) and their myeloid (mDCs) and plasmacytoid (pDCs) subsets is a less invasive procedure that is acquiring growing clinical relevance. Because dendritic cells (DCs) lack unique lineage markers, current methods that are based on 3- or 4-color assays do not allow multiparametric analysis of DC subsets. In this study a dedicated 6-color assay was developed. Methods: mDCs and pDCs were counted and characterized for the expression of activation/maturation markers by using a single-platform 6-color assay. Whole-blood samples from 20 healthy controls were directly stained with either CD80-FITC/CD40-PE/lineage-PerCP-Cy5.5/CD123-PE-Cy7/ CD11c-APC/HLA-DRAPC-Cy7 or CD86-FITC/CD83-PE/lineage-PerCP-Cy5.5/ CD123-PE-Cy7/CD11c-APC/HLA-DR-APC-Cy7 combination, in the presence of commercial fluorospheres. A dual-platform 3-color assay currently in use was run in parallel for comparison. Results: The 6-color assay provided mDCs and pDCs counts similar to counts obtained by the 3-color assay. Only the 6-color assay could show differential expression of activation markers by mDCs and pDCs, with pDCs expressing lower levels of costimulatory molecules and HLA-DR, but higher levels of CD83. Conclusions: The 6-color assay described here may be a sensitive tool for assessing possible variations in the number and features of mDCs and pDCs whose reciprocal balance is critical in understanding the more detailed orchestration of immune responses.

AB - Background: Flow cytometric analysis of peripheral blood dendritic cells (PBDCs) and their myeloid (mDCs) and plasmacytoid (pDCs) subsets is a less invasive procedure that is acquiring growing clinical relevance. Because dendritic cells (DCs) lack unique lineage markers, current methods that are based on 3- or 4-color assays do not allow multiparametric analysis of DC subsets. In this study a dedicated 6-color assay was developed. Methods: mDCs and pDCs were counted and characterized for the expression of activation/maturation markers by using a single-platform 6-color assay. Whole-blood samples from 20 healthy controls were directly stained with either CD80-FITC/CD40-PE/lineage-PerCP-Cy5.5/CD123-PE-Cy7/ CD11c-APC/HLA-DRAPC-Cy7 or CD86-FITC/CD83-PE/lineage-PerCP-Cy5.5/ CD123-PE-Cy7/CD11c-APC/HLA-DR-APC-Cy7 combination, in the presence of commercial fluorospheres. A dual-platform 3-color assay currently in use was run in parallel for comparison. Results: The 6-color assay provided mDCs and pDCs counts similar to counts obtained by the 3-color assay. Only the 6-color assay could show differential expression of activation markers by mDCs and pDCs, with pDCs expressing lower levels of costimulatory molecules and HLA-DR, but higher levels of CD83. Conclusions: The 6-color assay described here may be a sensitive tool for assessing possible variations in the number and features of mDCs and pDCs whose reciprocal balance is critical in understanding the more detailed orchestration of immune responses.

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KW - Plasmacytoid dendritic cells

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