A six-color flow cytometric assay for the analysis of peripheral blood dendritic cells.
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.
Laboratory of Immunology, Dipartimento di Scienze e Tecnologie Biomediche, Universita Degli Studi di Milano, Milan, Italy.
Stefania
S
Giannelli
Adriano
A
Taddeo
Pietro
P
Presicce
Maria Luisa
ML
Villa
Silvia
S
Della Bella
D000328:Adult; D015703:Antigens, CD / Q000032:analysis; D015415:Biomarkers / Q000032:analysis*; D001773:Blood Cells / Q000166:cytology* / Q000378:metabolism; D019070:Cell Lineage; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D008297:Male; D008875:Middle Aged; D055815:Young Adult
D015703:Antigens, CD; D015415:Biomarkers; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens