Intracellular Accumulation Assay for C. auris

Intracellular accumulation assay was performed according to the protocol by Srivastava and Ahmad [5 (link)], with minor modification. Briefly, C. auris isolates were grown overnight in YPD broth medium at 37 °C. Then, C. auris suspensions were incubated with FLU and FAR as previously described in Section 2.3. Post incubation, cells were pelleted (5000× g for 5 min) and washed in sterile PBS. Cells were re-suspended in sterile PBS (1 mL) supplemented with 2% glucose and 4 μM rhodamine 6G (Sigma, Taufkirchen, Germany) and incubated at 37 °C for 30 min. Cells were then washed twice with cold sterile PBS and 1 mL of fresh PBS was added to the pellet. Afterwards, 100 μL of suspension was pipetted into a flat-bottomed dark 96 well plate (Costar^®, Kennebunk, ME, USA) and fluorescence was measured with a fluorescence spectrophotometer (Tecan, Männedorf, Switzerland). The results were evaluated using MagellanTM Data Analysis Software and the intensity of fluorescence of samples was determined by relative fluorescence units (RFUs). The same suspension was immediately used for microscopy. Fluorescence was detected by an inverted fluorescence microscope (Zeiss, Jena, Germany) with excitation/emission spectra 525/548 nm. The pictures were captured by AxioCam ERc5s (Zeiss, Jena, Germany) and evaluated by software Motic Images Plus 3 (Hong Kong, China).