C. albicans vacuole integrity was assessed using the lipophilic vacuole membrane dye MDY-64 (Molecular probes, Fisher Scientific) following the manufacturer's recommended procedure. Briefly, cells were grown overnight on RPMI liquid medium with pH 4.5 at 30°C. Cells were pelleted and washed twice with fresh RPMI pH 4.5 and resuspended in the same medium at an OD595 of 0.1. VPA was added at different concentrations (10, 50, and 100 μg/ml). Cells were incubated for 2 h at 30°C under agitation. Aliquots were taken from VPA-treated and non-treated cultures and the MDY-64 was added at a final concentration of 10 μM. Cells were incubated at room temperature for 3 min prior to confocal microscopy visualization. Images were acquired with a 1.3-numerical-aperture (NA) 63x objective on a Leica DMI6000B inverted microscope connected to a Hamamatsu C9100-13 camera.
Pan1-green fluorescent protein (GFP), End3-GFP and LIFEACT-GFP (Epp et al., 2013 (link)) were visualized using confocal microscopy as follow: an overnight culture was diluted in SC supplemented with 10 or 50 μg/ml VPA to an OD595nm of 0.05 and grown for four generations at 30°C under agitation. Cells were imaged as described for the vacuole staining experiments.
Pan1-green fluorescent protein (GFP), End3-GFP and LIFEACT-GFP (Epp et al., 2013 (link)) were visualized using confocal microscopy as follow: an overnight culture was diluted in SC supplemented with 10 or 50 μg/ml VPA to an OD595nm of 0.05 and grown for four generations at 30°C under agitation. Cells were imaged as described for the vacuole staining experiments.
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