Quantifying β(1-3)-glucan in Candida cells

Wild-type and cho1ΔΔ cells were grown in YPD overnight (~16 h), and back diluted to fresh YPD with OD₆₀₀ of ~0.1. The cells were then shaken at 225 rpm for 3 h before 170 µM CBR-5884 or equivalent DMSO were added. The cells were further shaken for 30, 60, 120 min before staining with anti-β(1-3)-glucan antibody. The cells were stained as previously described (57 (link), 97 (link)) with the exception that goat anti-mouse antibody conjugated to Alexa Fluor 488 (Jackson ImmunoResearch) was used as the secondary antibody. For imaging, Candida cells were resuspended in 200 µL of Fluoromount-G mounting medium and visualized with a Leica SP8 white light laser confocal microscope. The pictures were taken through Leica Application Suite X office software and quantified as described above.