Yang for technical assistance. This work was supported by the National Heart, Lung, And Blood Institute (Award Number DP2HL117752) and the National Institute of Allergy and Infectious Diseases (Award Numbers R01AI103146 and R21AI130495) of the National Institutes of Health, as well as the Cardiovascular Research Institute and the Sandler Asthma Basic Research Center at the University of California, San Francisco. into mice for numerous functional studies, including depletion of basophils5 and rapid desensitization.6 Surprisingly, several results obtained with MAR-1-mediated basophil depletion have not been recapitulated in genetic models of basophil depletion5. These unexpected findings based on MAR-1 prompted us to reexamine the Aminopterin specificity of this antibody. We first sought to clarify whether moDCs express FcRI under inflammatory conditions. In mice treated intranasally with house dust mite extract (HDM) and examined 3 days later by flow cytometric analysis using the MAR-1 antibody, we were able to identify FcRI+ CD11c+ MHC-II+ CD11b+ inflammatory moDCs in the lungs and mediastinal lymph node (mLN), but we were Aminopterin surprised to find that these apparent FcRI+ moDCs were also present in FcRI-deficient mice (Fcer1aC/C, hereafter denoted as FcRI KO) (Fig 1A). Since the MAR-1 antibody labeled these cells in the absence of FcRI expression, we reasoned it would be more appropriate to call these MAR-1+ moDCs. As MAR-1+ moDCs have also been reported in viral contamination3, we next tested MAR-1 staining of moDCs in this setting. To mimic viral contamination, Aminopterin we administered intranasal poly I:C, a TLR3 ligand, and also detected MAR-1+ moDCs in the lungs. Similar to HDM exposure, we found that MAR-1+ moDCs could also be identified in poly I:C-treated FcRI KO mice comparable to control mice (Fig 1B). In contrast, blood basophils only stained with MAR-1 from control mice but not from FcRI KO mice (Fig 1C), confirming the genotype of the FcRI KO mice. We next considered whether the MAR-1 antibody may bind to other Fc receptors and stained cells from mice lacking the Fc receptor common gamma chain (Fcer1gC/C, hereafter denoted as FcRc KO), which is necessary for the normal surface expression of all activating Fc receptors. In FcRc KO mice, MAR-1 staining was greatly diminished on moDCs from the lungs and mLN after HDM-treatment (Fig 1D). Macrophages and monocytes express various Fc receptors but are not known to express FcRI in mice, yet one study had noted MAR-1 staining on monocytes.6 We tested whether MAR-1 would stain splenic red pulp macrophages, lung alveolar macrophages, peritoneal macrophages and blood monocytes. Indeed, MAR-1 stained all of these macrophages (Fig 1E) and a subset of blood Ly6CC and Ly6C+ monocytes in both wild-type and FcRI KO mice (Fig 1F). Similar to moDCs, MAR-1 staining was greatly reduced in these populations in FcRc KO mice. These observations imply that MAR-1 may be cross-reacting with other Fc receptors, thereby resulting in the detection of MAR-1+ cells in FcRI KO mice. As our findings above suggested that MAR-1 binds one of the activating Fc receptors, we next attempted to identify which Fc receptor(s) MAR-1 may bind to. In mice, the activating Fc receptors that bind IgG are FcRI (CD64), FcRIII (CD16) and FcRIV (CD16C2). F2RL1 We noticed that MAR-1 staining strongly correlated with FcRI (CD64) staining on moDCs (Fig 2A), and that MAR-1 staining correlated with FcRIV (CD16C2) staining on Ly6CC blood monocytes (Fig 2B). To definitively test whether MAR-1 was cross-reacting with FcRI (CD64) and FcRIV (CD16C2), we expressed specific Fc gamma receptors in a cell line and then stained with MAR-1 versus other Fc gamma receptor specific antibodies. Specifically, the Phoenix cell line, a altered 293T human embryonic.