ABSTRACTFc-C-type lectin receptor (Fc-CTLRs) probes are soluble chimeric proteins constituted of the extracellular domain of a CTLR fused with the constant fraction (Fc) of the human IgG. These probes are useful tools to study the interaction of CTLRs with their ligands, with applications similar to those of antibodies, often in combination with widely available fluorescent antibodies targeting the Fc fragment (anti-hFc). In particular, Fc-Dectin-1 has been extensively used to study the accessibility of β-glucans at the surface of pathogenic fungi. However, there is no universal negative control for Fc-CTLRs, making the distinction of specific versus nonspecific binding difficult. We describe here 2 negative controls for Fc-CTLRs: a Fc-control constituting of only the Fc portion, and a Fc-Dectin-1 mutant predicted to be unable to bind β-glucans. Using these new probes, we found that while Fc-CTLRs exhibit virtually no nonspecific binding toCandida albicansyeasts,Aspergillus fumigatusresting spores strongly bind Fc-CTLRs in a nonspecific manner. Nevertheless, using the controls we describe here, we were able to demonstrate thatA. fumigatusspores expose a low amount of β-glucan. Our data highlight the necessity of appropriate negative controls for experiments involving Fc-CTLRs probes.IMPORTANCEWhile Fc-CTLRs probes are useful tools to study the interaction of CTLRs with ligands, their use is limited by the lack of appropriate negative controls in assays involving fungi and potentially other pathogens. We have developed and characterized 2 negative controls for Fc-CTLRs assays: Fc-control and a Fc-Dectin-1 mutant. In this manuscript, we characterize the use of these negative controls with zymosan, a β-glucan containing particle, and 2 human pathogenic fungi,Candida albicansyeasts andAspergillus fumigatusconidia. We show thatA. fumigatusconidia nonspecifically bind Fc-CTLRs probes, demonstrating the need for appropriate negative controls in such assays.