Characterisation of myeloid receptor expression and interferon alpha/beta production in murine plasmacytoid dendritic cells by flow cytomtery.
This is a flow cytometric study of expression of a diverse set of myeloid receptors on murine splenic plasmacytoid dendritic cells (pDCs) and the description of a FACS based assay for measurement of interferon (IFN)alpha/beta. We have extended the known repertoire of PRR expressed on murine pDCs with the novel observation that they express Dectin-2 and contain intracellular MR. In addition, this is the first report of F4/80 and CD200 on murine pDCs. We have confirmed the observation by others that murine pDCs express CD200R, the lectin Dectin-1 and the scavenger receptor CD36. This report also details a flow cytometry-based protocol to measure the production of murine IFNalpha/beta by splenic pDC. Briefly, splenocytes can be stimulated with virus or a TLR9 agonist and IFNalpha/beta production by pDCs is detected following intracellular staining. pDCs are specifically identified by 120G8 staining at 6 h after stimulation with inactivated influenza virus, however the specificity of 120G8 for pDCs is reduced at times later than 12 h. This assay is suitable for use with splenocytes from some mouse strains (129/SvEv), but not others (C57BL/6J), probably due to C57BL6J producing insufficient amounts of IFN following stimulation to be detected by intracellular staining. However, IFN production by C57BL/6J splenocytes is readily detectable by bioassay. In addition to being more sensitive than intracellular staining, the bioassay is also more sensitive than an IFNalpha ELISA. The comparable sensitivities of these assays are often a critical determinant of the choice of assay and are an important consideration in experimental design.
Seeds
Rosalind E
RE
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
Gordon
Siamon
S
Miller
Joanna L
JL
D000818:Animals; D015703:Antigens, CD / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D000943:Antigens, Differentiation / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D018955:CD36 Antigens / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology; D016898:Interferon-alpha / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D016899:Interferon-beta / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D037181:Lectins, C-Type / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D010950:Plasma Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D012680:Sensitivity and Specificity
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018955:CD36 Antigens; D016898:Interferon-alpha; D037181:Lectins, C-Type; C445340:dectin-2, mouse; C036763:monocyte-macrophage differentiation antigen; D016899:Interferon-beta; C042739:antigens, CD200