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-DR-APC-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.
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-DR-APC-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.
Authors: S Della Bella; S Giannelli; V Cozzi; V Signorelli; M Cappelletti; I Cetin; M L Villa Journal: Clin Exp Immunol Date: 2011-02-24 Impact factor: 4.330
Authors: Pietro Presicce; Maria E Moreno-Fernandez; Celine S Lages; Kris I Orsborn; Claire A Chougnet Journal: Cytometry A Date: 2010-06 Impact factor: 4.355
Authors: Diana M Brainard; Edward Seung; Nicole Frahm; Annaiah Cariappa; Charles C Bailey; William K Hart; Hae-Sook Shin; Sarah F Brooks; Heather L Knight; Quentin Eichbaum; Yong-Guang Yang; Megan Sykes; Bruce D Walker; Gordon J Freeman; Shiv Pillai; Susan V Westmoreland; Christian Brander; Andrew D Luster; Andrew M Tager Journal: J Virol Date: 2009-05-06 Impact factor: 5.103
Authors: Claudia Carenza; Francesca Calcaterra; Ferdinando Oriolo; Clara Di Vito; Marta Ubezio; Matteo Giovanni Della Porta; Domenico Mavilio; Silvia Della Bella Journal: Front Immunol Date: 2019-06-11 Impact factor: 7.561