Fig. 3

Overview of the Fungi-Flow protocol. Step 1: Fungal sporulated forms are cultured overnight at 30 °C to obtain budding forms. They were stored at − 80 °C until use. Step 2: Antibody samples are diluted to normalized concentrations. Step 3: Fungi are incubated with serum. Step 4: After washing, fungi are incubated with secondary antibodies. Step 5: Data acquisition. Acquisition by flow cytometry permits discrimination of fungi according to size (FSC-A) and granularity (SSC-A). Step 6: Data analysis. Histograms show the median fluorescence intensity (MFI) for yeast forms of C. albicans after incubation with different dilutions of serum from a healthy donor followed by immunostaining with fluorescently-labeled anti-human IgG antibodies. This protocol can be applied to any culturable fungi. Relevant negative controls must be used to detect unspecific fluorescence. None = unstained fungal forms. NRA non-relevant antibody, IC isotype control