Defining human dendritic cell progenitors by multiparametric flow cytometry.
Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3-7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings.
Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.
Gaëlle
G
Breton
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA.
Jaeyop
J
Lee
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA.
Kang
K
Liu
Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.|Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
Michel C
MC
Nussenzweig
D001854:Bone Marrow Cells / Q000502:physiology; D002454:Cell Differentiation; D003713:Dendritic Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D013234:Stem Cells / Q000166:cytology*