Abstract
Here, we describe a protocol for a continuous flow system for C. albicans cultures growing adherent to a plastic surface. The protocol was adapted from a previous method established to simulate blood flow on endothelial cells (Wilson and Hube, 2010). The adapted protocol was used by us for the removal of molecules in C. albicans supernatants, especially farnesol, which accumulate over the time course of incubation and cannot be specifically depleted. The system used, however, allows various applications including the simulation of physiological flow conditions. Several example applications are given on the manufacturer’s website (https://ibidi.com/perfusion-system/112-ibidi-pump-system.html).
Keywords: Continuous flow, C. albicans, Quorum sensing, Farnesol, Filamentation, ibidi® pumps system
Background
Farnesol is a potent inhibitor of the yeast-to hypha transition (Hornby et al., 2001) in the human pathogenic fungus Candida albicans and also promotes the reversal to yeast growth from preformed filaments (Lindsay et al., 2012). The quorum sensing molecule (QSM) rapidly accumulates in the supernatant of a Candida albicans EED1 deletion strain and promotes the reverse morphogenesis and a hyphal maintenance defect of the mutant (Polke et al., 2017). As we were unable to block farnesol synthesis (Polke et al., 2017), we utilized the ibidi® pump system to remove the accumulating QSMs in the supernatant by uni-directional flow. Flow application, together with a constant medium exchange during the time course of incubation, significantly prolonged filamentation in the C. albicans eed1∆ mutant. This indicated the successful removal of QSM accumulates, and provided a direct link between hyphal maintenance and farnesol signaling in C. albicans. The system used for this protocol (ibidi® pump system) allows various applications under simulation of physiological flow conditions, and thus might be easily modified for other applications. Several example applications are given on the manufacturer’s website (https://ibidi.com/perfusion-system/112-ibidi-pump-system.html).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Microscopic evaluation of filamentation was performed using a ZEISS AxioVert inverted microscope and ZEN software.
Notes
Recipes
Acknowledgments
This work has been financially supported by the Studienstiftung des deutschen Volkes e.V. (to MP) and the Deutsche Forschungsgemeinschaft (DFG JA1960/1-1 to IJ). The protocol was adapted from Wilson and Hube (2010). We thank Dr. Duncan Wilson and Dr. Sascha Brunke for helpful suggestions on the ibidi® pump system setup.
References
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