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Peer-reviewed

A Flow-assay for Farnesol Removal from Adherent Candida albicans Cultures

从贴壁白色念珠菌培养物中去除法尼醇的流式实验

MP Melanie Polke
Ilse D. Jacobsen Ilse D. Jacobsen

发布: 2017年10月05日第7卷第19期 DOI: 10.21769/BioProtoc.2562 浏览次数: 8228

评审: Valentine V TrotterEmmanuel ZavalzaJose Thekkiniath

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Feb 2017

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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 (连续流动) search C. albicans (白色念珠菌) search Quorum sensing (群体感应) search Farnesol (法尼醇) search Filamentation (成丝) search ibidi® pumps system (ibidi® 泵系统) search

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

  1. Protective gloves and lab coat
  2. Pipette tips (TipOne) (STARLAB INTERNATIONAL, catalog numbers: S1111-6000 , S1113-1006 , S1110-3000 )
  3. µ-Slide VI0.4 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized (ibidi, catalog number: 80606 )
  4. Flask with absorber beads: dry beads (KC Trockenperlen® [Sorbead®] orange, BASF)
  5. Micro-tubes, 1.5 ml (SARSTEDT, catalog number: 72.690.001 )
  6. 0.2 μm sterile filters (Minisart 0.2) (SARSTEDT, catalog number: 83.1862.001 )
  7. Syringe Injekt® 10 ml/Luer Lock Solo, sterile (B. Braun Medical, catalog number: 4606728V-02 )
  8. 50 ml Falcon tubes (SARSTEDT, catalog number: 62.547.254 )
  9. 10 ml pipette, graduated, sterile (Greiner Bio One International, catalog number: 607180 )
  10. Petri dishes (Greiner Bio One International, catalog number: 633180 )
  11. Disposal bags (Carl Roth, catalog number: E706.1 )
  12. Steam Indicator Tape 3M (ComplyTM, 3M, catalog number: 1322-18MM )
  13. Perfusion Set Blue (ibidi, catalog number: 10961 )
  14. Filter/Reservoir set (10 ml, sterile) (ibidi, catalog number: 10971 )
  15. Candida albicans strains of interest (the system was established using SC5314 and the respective EED1 deletion mutant, see Polke et al., 2017)
  16. RPMI1640 medium [(+)L-glutamine, (+)phenol red, unbuffered] (Thermo Fisher Scientific, GibcoTM, catalog number: 21875034 )
  17. Fermacidal D2® (2%) (LABOTECT, catalog number: 15101 )
  18. Roti®-Histofix 4% (Carl Roth, catalog number: P087.5 )
  19. Glycerol, ROTIPURAN®, water-free (Carl Roth, catalog number: 3783.2 )
  20. D(+)-Glucose, water-free (Carl Roth, catalog number: HN06.4 )
  21. BactoTM peptone (BD, BactoTM, catalog number: 211677 )
  22. Yeast extract, micro-granulated (Carl Roth, catalog number: 2904.1 )
  23. Agar-agar, Kobe I (Carl Roth, catalog number: 5210.4 )
  24. Sodium chloride (NaCl) (Carl Roth, catalog number: 9265.2 )
  25. Disodium phosphate (Na2HPO4·2H2O) (Carl Roth, catalog number: T877.1 )
  26. Monopotassium phosphate (KH2PO4) (Carl Roth, catalog number: 3904.1 )
  27. Ethanol denatured ≥ 99.8% (Carl Roth, catalog number: K928.4 )
  28. 30% glycerin solution (see Recipes)
  29. 20% D(+)-glucose solution (see Recipes)
  30. YPD broth (see Recipes)
  31. YPD agar medium (see Recipes)
  32. 10x PBS (see Recipes)
  33. 1x PBS (see Recipes)
  34. 70% ethanol (see Recipes)

Equipment

  1. Milli-Q® integral water purification system for ultrapure water (deionized water) (Merck, model: Milli-Q® Integral )
  2. Infors HT, Multitron Standard shaking incubator, Version 2 (Infors, model: Multitron Standard )
  3. BINDER cooling incubator (series: APT.line®KB, BINDER, model: KB 53 ; 30 °C)
  4. BINDER CO2 incubator (series APT.line®CB, BINDER, model: CB 220 ; 37 °C)
  5. Glass flasks 25 ml and 250 ml (Schott, DURAN, Germany)
  6. Pipette set 0.2 μl-1,000 μl (Gilson, model: PIPETMAN® P, P2 , P10 , P20 , P100 , P200 and P1000 )
  7. Tabletop centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HereausTM PicoTM 21 )
  8. Mid bench centrifuge (Sigma Laborzentrifugen, model: SIGMA 3-18K )
  9. Vortexer (Scientific Industries, model: Vortex-Genie 2 )
  10. Neubauer improved, cell counting chamber 0.0025 mm2 (Marienfeld-Superior, catalog number: 0640030 )
  11. Biosafety cabinet (NuAire, model: NU-480-400E )
  12. Ibidi® pump system including ibidi pump, fluidic unit, perfusion set, notebook, PumpControl software (ibidi, catalog number: 10902 )
  13. ZEISS inverted microscope (ZEISS, model: Axio Vert.A1 )
  14. UV Crosslinker (Vilber, model: Bio-Link 254 )
  15. Autoclave (for example: SHP Steriltechnik, model: Laboklav 135 MSLV )

Software

  1. Computer equipped with ZEISS ZEN software (Blue edition, 2012)
  2. Computer with GraphPad Prism 5 software

Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Polke, M. and Jacobsen, I. D. (2017). A Flow-assay for Farnesol Removal from Adherent Candida albicans Cultures. Bio-protocol 7(19): e2562. DOI: 10.21769/BioProtoc.2562.

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分类

微生物学 > 微生物信号传导 > 群体感应

细胞生物学 > 细胞信号传导 > 群体感应

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