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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   

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Molecular Microbiology
Feb 2017

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

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

  1. Growth of Candida albicans cells and germ tube induction
    1. Streak respective C. albicans strains from 30% glycerol stocks (see Recipes) onto YPD agar (see Recipes) and grow for 2 d at 30 °C in an incubator.
    2. Pick a single colony from each strain and re-streak onto a fresh YPD agar plate. Grow for 2 d at 30 °C in an incubator.
    3. Pick a single colony from each strain and inoculate in 10 ml YPD medium (see Recipes) in a 25 ml glass flask. Grow overnight at 30 °C with vigorous horizontal shaking (180 rpm) in a shaking incubator.
    4. Dilute the o/n culture 100-fold (100 μl) in fresh 10 ml YPD medium and grow overnight (approximately 20 h) in a 25 ml glass flask at 30 °C with horizontal shaking (180 rpm) to semi-synchronize the growth of all strains.
    5. On the day of the experiment harvest 1 ml of the overnight culture of each strain into a 1.5 ml micro-tube and centrifuge at 10,000 x g for 1 min at room temperature in a tabletop centrifuge to collect cells. Remove the supernatant and resuspend the cells in 1 ml sterile PBS (see Recipes) to wash the yeast.
    6. Repeat the washing step described above twice.
    7. After the last wash, resuspend the yeast in 1 ml PBS.
    8. Prepare an appropriate dilution of cells for determination of the total cell number in suspension; for the wild type C. albicans strain, a 20 h culture prepared as described above, a 100-fold dilution of the initial culture is usually appropriate.
    9. Apply 10 μl of the dilution onto a Neubauer cell counting chamber and determine the cell number per ml using a microscope following the manufacturer’s instructions.
    10. Seed 1 x 105 cells/ml into 50 ml RPMI1640 medium (pre-warmed at 37 °C) in a 250 ml glass flask and incubate with shaking (180 rpm) for 30 min at 37 °C to induce germ tube formation.
  2. Equilibrate the RPMI1640 medium and the materials (perfusion set) before the experiment at 37 °C and 5% CO2 (incubation conditions during the flow assay); this is best performed overnight.
    Note: Prepare the equipment of the ibidi® pump system and the incubation setup before harvesting the C. albicans cultures to be ready to start when the germ tubes are formed (see above). The preparation of the fluidic unit and the perfusion set must occur under sterile conditions. The µ-slide must be prepared and connected to the pump system before the complete unit can be placed in the incubator. The assembly of the system must be performed in a biosafety cabinet.
  3. Prepare the perfusion set (see Figure 1)


    Figure 1. Fluidic unit with perfusion set. The two reservoirs have been mounted to the holder of the fluidic unit. Prepare the unit under a clean bench for sterility. Fluidic unit (1), reservoir 10 ml (2), 0.2 μm sterile filter (3), tubing (4), valve (5) and tube-endings with Luer locks connected by the Luer Lock Coupler (6).

    1. Attach the reservoirs of the perfusion set to the fluidic unit; close/connect the ends of the tubings with the included Luer Lock Coupler, and remove the mounted filters capping the reservoirs.
    2. Add ~10 ml of equilibrated RPMI1640 medium to each of the reservoirs (Figure 2A). The medium will spread across the tubing system.
    3. To remove remaining air bubbles in the tubes, open the Luer Lock Coupler connection (medium will leak from the tube so make sure to collect it in a Falcon tube or to soak it on a paper sheet). Use a sterile plug from a 10 ml syringe to apply pressure on the medium by inserting the plug into the perfusion set reservoirs. Remove all air bubbles by pushing medium through the system (Figures 2B and 2C). Avoid letting the tubing system run dry as this will cause new air bubbles to arise.
    4. When all air bubbles are removed, sterilize the Luer connection at both ends with 2% Fermacidal (or another suitable disinfectant) and reconnect the ends of the tubings with the Luer Lock Coupler. Fill up each reservoir with equilibrated RPMI1640 medium to 5-5.5 ml (Figure 2D; the total working volume of the Blue Perfusion set system is 11.3 ml).
    5. Close the reservoirs by mounting the filters (Figure 2E). The filters are necessary to avoid contamination with air-borne microbes through the tubing system during pressure application by the pump.
    6. Place the prepared fluidic unit, including the prepared perfusion set, into the CO2 incubator (37 °C 5% CO2) for pre-warming until the beginning of the experiment (for at least 1 h if the media was pre-warmed; the unit can be set up the day before the actual experiment).


      Figure 2. Preparation of the fluidic unit and removal of air bubbles in the system. Fill both reservoirs with medium (A). The medium will spread through the tubings, but air bubbles remain. To remove air bubbles, use a 10 ml syringe plunge to apply weak pressure in both reservoirs (B and C). Make sure to collect leaking (sterile) medium in a tube or on paper sheets (D). When all air bubbles are removed, disinfect the Luer connector and tube endings, reconnect the tube endings with the Luer Lock Coupler, and close the reservoirs with the supplied filters.

  4. Set up the pump by connecting it to the fluidic unit, power supply, and computer and adjust the settings in the ibidi® pump system software:
    1. Connect the pump to the notebook and connect all tubings as indicated in Figure 3. Note that a flask with absorber beads must be placed between the pump and the fluidic unit to remove the moisture generated in the tubings during incubation in the humidified incubator chamber.
    2. Start the software for the ibidi® pump.
    3. For uni-directional flow with 2 ml/min flow rate choose the parameters as given in Figure 4.


      Figure 3. Setup of the computer and pump system. The parts of the system shown in (A) remain on the bench, while the fluidic unit is placed within the CO2 incubator. The connections between the pump, absorber, and computer are indicated in B: The pump (1) is connected to the power supply (2) and the computer (3). The tubing for applying pressure is mounted on the pump (4), and connected to the absorber bottle (5). From the absorber bottle, the long tube (6) will be connected with the fluidic unit in the incubator to apply pressure on the medium in the reservoirs. The pump contains several channels for connection to fluidic units (C, indicated by arrow). Up to four fluidic units can be controlled by one pump. Choose the right connection when setting up the experiment in the software (see Figure 4).


      Figure 4. ibidi® pump control software setup for a flow rate of 2 ml/min. A. Open the software and open the Scheduler Wizard Overview by clicking on the button indicated in A1. A new window will open. In our application (Polke et al., 2017), the parameters as indicated in A2 were used: Approx. Flow 2,00 ml/min; Suggested Pressure 13,87 mbar; Continuous Valves ‘2’; Continuous Cycle Time 45,00 sec; Oscillating Valves: ‘not set’; Oscillating Cycle Time: ‘not set’; Duration: 86,400 sec. Slide Selection: µ-slide VI0.4; Perfusion Set Selection: 15 cm, ID 0.8 mm (blue); Viscosity 0,01. When you finished setting up the parameters, select ‘Create Schedule’ as indicated by A3. B. The parameters will appear in the Pump Control; at this point, software and pump are ready for use. To connect the fluidic unit/perfusion to the pump (following step 7 of this protocol), choose the appropriate pump channel (B1, also see Figure 3), apply pressure (B2) and start the flow (B3).

  5. Collect the C. albicans germ tubes and seed onto ibidi μ-slide
    1. To collect germ tubes, centrifuge the culture prepared according to step 1j in a 50 ml Falcon tube at 4,000 x g for 10 min to 20 min at room temperature. While collection of germ tubes can be tricky and sometimes requires long centrifugation steps, in our hands the germ tubes at 30 min post hypha-induction can be successfully collected by centrifugation at 4,000 x g for 10 min at room temperature.
    2. Carefully remove the supernatant by aspirating using a pipette (as the cell pellet may be ‘fluffy’ it easily gets lost when the supernatant is decanted). Resuspend cells in 10 ml fresh (pre-warmed to 37 °C) RPMI1640 to get a final cell density of 5 x 105 cells/ml. For our experiments cell density was not critical and we therefore assumed that no cells were lost during supernatant removal; for other applications, however, it might be necessary to re-count the cells before final resuspension to obtain a more accurate cell density.
    3. Carefully vortex the cell suspension and seed the C. albicans cells on the ibidi μ-slide VI0.4 by adding 30 μl to each channel to be used (e.g., one for flow condition, one for static condition; see Figure 5). The final cell number in the channel (0.6 cm2 of growth area) is 1.5 x 104 cells/ml.


      Figure 5. Cell seeding into the ibidi μ-slide VI0.4. Carefully pipette the cell suspension into the small reservoirs of the μ-slide under sterile conditions (A). 30 μl is enough to fill the cell channel, if you wish you can also apply more medium. Close the lid (B), and place the μ-slide in an incubator (37 °C 5% CO2 humidified chamber) for 20 min to allow adherence of cells.

    4. Close the channels with the included lid and incubate for 20 min at 37 °C and 5% CO2 to allow adhesion of germ tubes to the plastic surface. During the flow, all non-adherent cells would be washed off and accumulate in the medium in the reservoirs.
  6. Wash cells and connect ibidi μ-slide to the perfusion set
    1. Following adhesion, carefully remove the medium from the channels, wash cells once by adding 150 μl pre-warmed RPMI1640 medium and aspirating again. This will remove most non-adherent cells. Avoid vigorous pipetting as this might detach cells.
    2. For the cells grown under static conditions, apply 120 μl pre-warmed RPMI1640 medium and close the channel with the lid.
    3. For cells grown under flow conditions, add ~200 μl pre-warmed RPMI1640 medium to the appropriate channel (the channel should be completely filled with fluid [according to Figure 6A]).
    4. Remove the fluidic unit, including the perfusion set, from the incubator, and place it under a clean bench. Open the Luer Lock Coupler and rapidly attach the Luer connections to the small reservoirs on each end of the channel (see Figure 6). Avoid medium spilling from the tubes. This is easily done when the tubing is temporarily closed with a clip, as visible in Figure 6. The ibidi μ-slide is now prepared for incubation, and can be fixed on the fluidic unit as indicated in Figure 6D.


      Figure 6. Connecting the ibidi μ-slide VI0.4 to the perfusion set (under sterile conditions). Close the tubes with a standard clip to avoid medium leakage from the tubes during connection. Remove the Luer Lock Coupler and attach each end of the tubes to one of the small reservoirs (A). Make sure that the channel is completely full of fluid to avoid introducing air bubbles into system. Cover the cells grown under static conditions (B). Place the μ-slide on the designated space on the fluidic unit (C) and fix the slide with adhesive tape to avoid movement or slipping during transport (D). Cells are then grown under flow (E1) or static conditions (E2), respectively.

    5. Connect the tubes of the Perfusion set in correct arrangement with the valve set: During the run/flow, medium from one reservoir is pumped through the tubes and the μ-slide channel into the other reservoir and vice versa. In order to allow uni-directional flow in the channel, the fluidic unit contains two different valves which are switched upon pumping in one or the other direction. A nice description of the principle is given on the manufacturer’s website (https://ibidi.com/perfusion-system/112-ibidi-pump-system.html) and in (Wilson and Hube, 2010). To allow the correct switching of valves, the tubes have to be fixed in a criss-crossed order (see Figures 7A and 8).


      Figure 7. Flow application. Connect the tubings with the valve (A). Make sure to place them in the right order to allow a uni-directional flow in the μ-channel. Set-up in a criss-crossed order as indicated in the picture. The arrows indicate the markings which help to place the branched tubes in the right order. Connect the fluidic unit to the pump, and place the fluidic unit in the incubator (B). Apply flow and check for correct pumping between the reservoirs. Incubate for 6 h (or the desired time).


      Figure 8. Principle of tubing connections and flow. A. Set up of the tubing system. Note that from the two tubes that originate from each reservoir, one each are connected. B. Principle of the valve system and flow directions. Within the pump, the tubes from the two reservoirs that are coupled after the pump are placed in the same valve.

  7. Place the Fluidic unit including the Perfusion set and ibidi μ-slide in the incubator (Figure 7B).
  8. Apply flow by starting the pump via the software. Check if the flow is correct. No unbalanced medium flow between the reservoirs should occur, as this would result in aspiration of air over the time. Unbalanced medium flow can be easily detected by checking the amount of media in the two reservoirs: It leads to non-uniform medium distribution in the two reservoirs, which does not decrease but increase after starting the flow. If the flow appears to run smoothly, close the incubator door. While doing so, avoid crippling of the tubes and cables as kinks will hamper the media flow. Incubate the yeast under static/flow conditions for the desired time.
  9. To avoid accumulation of quorum sensing molecules and waste products in the medium of the reservoirs remove the fluidic unit from the incubator each hour during the experiment: Pause the medium flow and detach the Fluidic unit from the pump and computer. Quickly open the reservoirs under sterile conditions, remove ~10 ml (of ~11.3 ml total volume in the reservoirs) of medium. Do not aspirate too much medium to avoid air bubble formation in the tubes. Immediately replace the removed liquid with fresh RPMI1640 medium (10 ml, pre-warmed to 37 °C, equilibrated with 5% CO2).
    Note: Do NOT remove the ibidi μ-slide from the fluidic unit during medium exchange!
  10. At the end of the incubation, remove the fluidic unit from the incubator, detach the μ-slide from the tubes of the Perfusion set, and remove the medium from each channel.
  11. Carefully flush each channel containing cells three times each with 150 μl 1x PBS. Then, add 120 μl Histofix (or 4% paraformaldehyde) to fix the cells for 1 h at room temperature. Replace Histofix with 120 μl 1x PBS for microscopy.

Data analysis

Microscopic evaluation of filamentation was performed using a ZEISS AxioVert inverted microscope and ZEN software.

  1. Start the software.
  2. Start the microscope, place ibidi chamber on the micro slide holder and adjust.
  3. Make sure to choose the right magnification in the software for microscopy. You find the respective selection (for objective, zoom and Camera Adapter) in the settings ‘Microscope Components’. This is important for the software to calculate the correct μm length from pixels.
  4. Take pictures. Make sure to choose several random spots throughout the whole channel for quantification of filament length during microscopy. In our study (Polke et al., 2017) we analyzed the length of 70 random cells in each channel per replicate (five replicates in total).
  5. For analysis of filament length, set the ‘Scaling’ to ‘theoretic’ and choose the desired unit (μm are usually appropriate) in the ‘Scaling’ settings.
  6. To measure filament length, select the tool ‘active curve’ in the folder ‘Graphics’. This allows an exact filament length determination. This is important as hyphae are not always growing straight, but may be curvy (see Figure 9), which would falsify the filament length determination when measured in a straight line. For filament length measurement, leave out the mother yeast, but include budding yeast which have not yet separated (pseudohyphae; see Figure 9).


    Figure 9. Filament length measurement of C. albicans eed1∆ cells grown under flow (left) or static (right) conditions using ZEN software (ZEISS). The length of each filament was measured using the active curve tool of the ZEN software (Blue edition, ZEISS). The measured part of the filaments is indicated by the red line.

  7. Measure the length of filaments grown under static and flow conditions. Alternatively, filament induction can be determined by counting filament numbers in relation to the total cell number.
  8. In our study, the filament lengths from static or flow conditions were analyzed statistically by the non-parametric Mann-Whitney test using GraphPad Prism 5 software (see Figure 10).


    Figure 10. Quantification of the filament length of a C. albicans EED1 deletion strain (eed1∆) grown under flow or static conditions (graph taken from [Polke et al., 2017]). Filament lengths from static or flow conditions were analysed statistically by the non-parametric Mann-Whitney test using GraphPad Prism 5 software. The numbers in the bars indicate the average filament length from 70 cells in three independent biological experiments under the given growth conditions.

Notes

  1. Some of the ibidi® pump system materials can be sterilized by autoclaving and reused for the experimental setup. These include the tubings, and the attached reservoirs, but not the mounted filters. For sterilization, place the tubings and reservoirs in a heat stable autoclaving bag (e.g., disposal bag), close the bag using Steam Indicator Tape and sterilize in a standard laboratory autoclave at 121 °C for 20 min. For sterilization of the filters, wipe the filters several times with 70% ethanol and subsequently sterilize under UV light in a UV-crosslinker (120 mJ per cm2) or a comparable UV device for 15 min. However, we recommend not to reuse tubings and reservoirs for more than three times.
  2. In the flow assay system used for the experiments in our publication, only one fluidic unit (ibidi) was available and installed to generate a uni-directional flow on cells. However, one ibidi µ-slide VI0.4 has six channels for cell seeding. Thus, it would be possible to grow more than one sample under flow conditions if several fluidic units are installed. Always include a static sample as control on the slide if applicable to your scientific question.
  3. While we used the ibidi® pump system to generate a uni-directional flow to wash of accumulating QSMs in the cell culture supernatant, the system may also be used to simulate flow on a biofilm surface, to simulate blood flow (Wilson and Hube, 2010) and for many more applications. A detailed transcription on the principal of the ibidi® pump system, the uni directional flow assurance and more applications using the pump system are provided on the manufacturer’s homepage (http://ibidi.com/).
  4. Other flow systems from other manufacturers can of course be used for this type of experiments. Important aspects when choosing a system for the experiment described here are the ease with which medium can be removed from the system, and the total medium volume in relation to the channel volume, as this determines the dilution rate. For the ibidi® pump system, for example, also bigger reservoir sets (50 ml) are available.

Recipes

  1. 30% glycerin solution (100 ml)
    30 ml glycerol, ROTIPURAN®, water-free
    Add 70 ml deionized water
    Autoclave
  2. 20% D(+)-glucose solution (500 ml)
    100 g D(+)-glucose solution, water-free
    Add deionized water to 500 ml
    Autoclave
  3. YPD broth (for 500 ml)
    2% (w/v) (10 g) BactoTM peptone
    1% (w/v) (5 g) yeast extract, micro-granulated
    Add deionized water to 450 ml, autoclave
    Add 50 ml sterile 20% D(+)-glucose solution (final concentration 2%)
  4. YPD agar medium (500 ml)
    2% (w/v) (10 g) BactoTM peptone
    1% (w/v) (5 g) yeast extract, micro-granulated
    2% (w/v) (10 g) D(+)-glucose
    2% (w/v) Agar-agar, Kobe I
    Autoclave
  5. 10x PBS
    1.5 M NaCl
    100 mM Na2HPO4·2H2O
    12 mM KH2PO4
    Adjust pH to 7.4
    Autoclave
  6. 1x PBS
    100 ml 10x PBS, add 900 ml sterile water
  7. 70% ethanol (for 1 L)
    Mix 700 ml ethanol (denatured, ≥ 99.8%) and 300 ml deionized water

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

  1. Hornby, J. M., Jensen, E. C., Lisec, A. D., Tasto, J. J., Jahnke, B., Shoemaker, R., Dussault, P. and Nickerson, K. W. (2001). Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl Environ Microbiol 67(7): 2982-2992.
  2. Lindsay, A. K., Deveau, A., Piispanen, A. E. and Hogan, D. A. (2012). Farnesol and cyclic AMP signaling effects on the hypha-to-yeast transition in Candida albicans. Eukaryot Cell 11(10): 1219-1225.
  3. Polke, M., Sprenger, M., Scherlach, K., Alban-Proano, M. C., Martin, R., Hertweck, C., Hube, B. and Jacobsen, I. D. (2017). A functional link between hyphal maintenance and quorum sensing in Candida albicans. Mol Microbiol 103(4): 595-617.
  4. Wilson, D. and Hube, B. (2010). Hgc1 mediates dynamic Candida albicans-endothelium adhesion events during circulation. Eukaryot Cell 9(2): 278-287.

简介

在这里,我们描述了用于连续流系统的协议。 白色念珠菌生长粘附在塑料表面上的培养物。 该方案根据建立以模拟内皮细胞上的血液流动的先前方法改编(Wilson和Hube,2010)。 我们使用适应方案去除C中的分子。 白色念珠菌上清液,特别是法呢醇,其在孵育的时间过程中积累并且不能被特异性地耗尽。 然而,所使用的系统允许各种应用,包括模拟生理流动条件。 制造商网站上给出了几个示例应用程序(https://ibidi.com/perfusion-system/112-ibidi-pump-system.html).
【背景】法尼醇是人类致病真菌白色念珠菌中的酵母 - 菌丝转移(Hornby等人,2001)的有效抑制剂,并且还促进了酵母生长的逆转预制长丝(Lindsay等人,2012)。群体感知分子(QSM)快速积聚在白念珠菌EED1缺失株的上清液中,并促进突变体(Polke等人)的反向形态发生和菌丝维持缺陷>,2017)。由于我们无法阻止法呢醇的合成(Polke等人,2017),我们使用了ibidi ®泵系统,通过单向去除上清液中累积的QSMs流。流动应用以及在孵育期间的恒定的培养基更换在C中显着延长了丝状化。白色念珠菌eed1Δ突变体。这表明QSM积累的成功去除,并且提供了在C中的菌丝维持和法呢醇信号之间的直接联系。白色假丝酵母。用于此协议(ibidi ®泵系统)的系统允许在生理流动条件的模拟下的各种应用,因此可能容易地针对其他应用进行修改。制造商网站上给出了几个示例应用程序(https://ibidi.com/perfusion-system/112-ibidi-pump-system.html).

关键字:连续流动, 白色念珠菌, 群体感应, 法尼醇, 成丝, ibidi® 泵系统

材料和试剂

  1. 防护手套和实验室外套
  2. 移液器吸头(TipOne)(STARLAB INTERNATIONAL,目录号:S1111-6000,S1113-1006,S1110-3000)
  3. μ-Slide VI 0.4 ibiTreat:#1.5聚合物盖玻片,组织培养处理,灭菌(ibidi,目录号:80606)
  4. 带吸收珠的烧瓶:干燥珠(KC Trockenperlen [Sorbead ],橙,BASF)
  5. 微管1.5 ml(SARSTEDT,目录号:72.690.001)
  6. 0.2微米无菌过滤器(Minisart 0.2)(SARSTEDT,目录号:83.1862.001)
  7. 注射器Injekt ® 10 ml / Luer Lock Solo,无菌(B.Braun Medical,目录号:4606728V-02)
  8. 50ml Falcon管(SARSTEDT,目录号:62.547.254)
  9. 10 ml移液器,分级,无菌(Greiner Bio One International,目录号:607180)
  10. 培养皿(Greiner Bio One International,目录号:633180)
  11. 处理袋(Carl Roth,目录号:E706.1)
  12. 蒸汽指示胶带3M(Comply TM ,3M,目录号:1322-18MM)
  13. 灌注组蓝(ibidi,目录号:10961)
  14. 过滤器/储存器组(10ml,无菌)(ibidi,目录号:10971)
  15. 感兴趣的菌株(使用SC5314和相应的EED1缺失突变体建立系统,参见Polke等人,2017)
  16. RPMI1640培养基[(+)L-谷氨酰胺,(+)酚红,无缓冲液](Thermo Fisher Scientific,Gibco TM,目录号:21875034)
  17. Fermacidal D2 ®(2%)(LABOTECT,目录号:15101)
  18. Roti ® -Histofix 4%(Carl Roth,目录号:P087.5)
  19. 甘油,ROTIPURAN ®,无水(Carl Roth,目录号:3783.2)
  20. D(+) - 葡萄糖,无水(Carl Roth,目录号:HN06.4)
  21. Bacto TM 蛋白胨(BD,Bacto TM,目录号:211677)
  22. 酵母提取物,微粒(Carl Roth,目录号:2904.1)
  23. 琼脂,神户一(Carl Roth,目录号:5210.4)
  24. 氯化钠(NaCl)(Carl Roth,目录号:9265.2)
  25. 磷酸二钠(Na 2 HPO 4•2H 2 O)(Carl Roth,目录号:T877.1)
  26. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:3904.1)
  27. 乙醇变性≥99.8%(Carl Roth,目录号:K928.4)
  28. 30%甘油溶液(参见食谱)
  29. 20%D(+) - 葡萄糖溶液(参见食谱)
  30. YPD肉汤(见食谱)
  31. YPD琼脂培养基(见食谱)
  32. 10x PBS(参见食谱)
  33. 1x PBS(见食谱)
  34. 70%乙醇(见食谱)

设备

  1. Milli-Q ®超纯水(去离子水)的积分净水系统(默克,型号:Milli-Q 积分)
  2. Infors HT,Multitron标准振荡培养箱,版本2(Infors,型号:Multitron Standard)
  3. BINDER冷藏培养箱(系列:APT.line ® KB,BINDER,型号:KB 53; 30°C)
  4. BINDER CO 2 培养箱(系列APT.line CB,BINDER,型号:CB 220; 37℃)
  5. 玻璃瓶25毫升和250毫升(Schott,DURAN,德国)
  6. 移液器设置0.2μl-1,000μl(Gilson,型号:PIPETMAN P / P,P2,P10,P20,P100,P200和P1000)
  7. 台式离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Hereaus TM Pico TM 21)
  8. 中型台式离心机(Sigma Laborzentrifugen,型号:SIGMA 3-18K)
  9. Vortexer(科学工业,型号:Vortex-Genie 2)
  10. Neubauer改进,细胞计数室0.0025 mm 2(Marienfeld-Superior,目录号:0640030)
  11. 生物安全柜(NuAire,型号:NU-480-400E)
  12. Ibidi ®泵系统,包括ibidi泵,流体单元,灌注套件,笔记本,PumpControl软件(ibidi,目录号:10902)
  13. 蔡司倒置显微镜(ZEISS,型号:Axio Vert.A1)
  14. 紫外线交联剂(Vilber,型号:Bio-Link 254)
  15. 高压灭菌器(例如:SHP Steriltechnik,型号:Laboklav 135 MSLV)

软件

  1. 配有ZEISS ZEN软件的电脑(Blue edition,2012)
  2. 带GraphPad Prism 5软件的计算机

程序

  1. 白念珠菌细胞和胚管诱导的生长
    1. 条纹分别为C.来自30%甘油储备(见食谱)的白色念珠菌菌株在YPD琼脂上(参见食谱),并在孵育器中在30℃下生长2天。
    2. 从每个菌株中挑取一个菌落并重新连接到新鲜的YPD琼脂平板上。在孵化器中在30℃下生长2天。
    3. 从每个菌株中挑取一个菌落,并在25 ml玻璃瓶中接种到10ml YPD培养基(参见食谱)中。在振荡的培养箱中,在30°C下生长过夜,同时进行剧烈的水平振荡(180rpm)
    4. 在新鲜的10ml YPD培养基中稀释100倍(100μl)的o / n培养物,并在30℃的玻璃烧瓶中在30℃下用水平摇动(180rpm)生长过夜(约20小时),使生长的所有菌株
    5. 在实验当天,将每个菌株的1ml过夜培养物收集到1.5ml微管中,并在室温下以10,000×g离心1分钟,在台式离心机中收集细胞。取出上清液,将细胞重悬于1 ml无菌PBS(见食谱),以洗涤酵母
    6. 重复上述两次洗涤步骤。
    7. 最后一次洗涤后,将酵母重悬于1 ml PBS中
    8. 准备适当稀释的细胞以确定悬浮液中的总细胞数;对于野生型 C。白色念珠菌菌株,如上所述制备的20小时培养基,初始培养物的100倍稀释度通常是适当的。
    9. 将10μl稀释液加入到Neubauer细胞计数室中,并按照制造商的说明书使用显微镜测定细胞数/ ml。
    10. 将种子1×10 5个细胞/ ml加入到250ml玻璃烧瓶中的50ml RPMI1640培养基(在37℃下预热)中并在37℃下振摇(180rpm)孵育30分钟诱导胚管形成
  2. 在37℃和5%CO 2(实验过程中的孵育条件)下,实验前平衡RPMI1640培养基和材料(灌注组);这最好在一夜之间完成。
    注意:准备ibidi ® 泵系统的设备和孵化设置,然后收获白色念珠菌培养物即可当胚芽管形成时开始(见上文)。流体单元和灌注装置的制备必须在无菌条件下进行。必须准备μ型滑块并将其连接到泵系统,才能将完整的单元放置在培养箱中。系统的组装必须在生物安全柜内进行。
  3. 准备灌注组(见图1)


    图1.带有灌注液的流体单元。 两个储存器已经安装到流体单元的支架上。准备单位在一个干净的工作台上不育。流体单元(1),容器10 ml(2),0.2μm无菌过滤器(3),管道(4),阀门(5)和管路端部,Luer锁定器由Luer Lock Coupler(6)连接。 >
    1. 将灌注装置的储液器连接到流体装置;使用随附的Luer Lock Coupler封闭/连接管道的两端,并卸下已安装的过滤器,以覆盖水箱。
    2. 将平衡的RPMI1640培养基加入到每个储存器中(图2A)。介质将分布在管道系统中。
    3. 为了清除管中剩余的气泡,请打开Luer Lock Coupler连接(介质将从管中泄漏,以确保将其收集在Falcon管中或将其浸泡在纸张上)。使用10ml注射器的无菌塞,通过将塞子插入灌注液储存器来对介质施加压力。通过将介质推入系统中去除所有气泡(图2B和2C)。避免让管道系统干燥,这样会引起新的气泡。
    4. 当所有气泡都被去除时,两端的Luer连接用2%Fermacidal(或其他合适的消毒剂)消毒,并用Luer Lock Coupler重新连接管道的两端。用平衡的RPMI1640培养基将每个储存器填充至5-5.5ml(图2D; Blue Perfusion系统的总工作体积为11.3ml)。
    5. 通过安装过滤器关闭油箱(图2E)。过滤器是必要的,以避免在通过泵的压力施加期间通过管道系统污染空气传播的微生物。
    6. 将制备好的流体单元(包括准备好的灌注装置)置于CO 2培养箱(37℃5%CO 2)中,以便预热直到实验开始(如果媒体预热了至少1小时,该单位可以在实际实验前一天设置)。


      图2.制备流体单元并清除系统中的气泡。 用中等(A)填充两个水库。介质将通过管道扩散,但气泡仍然存在。为了去除气泡,请使用10ml注射器插入在两个储存器(B和C)中施加弱压。确保收集管或纸张上的泄漏(无菌)培养基(D)。当所有气泡被去除时,消毒Luer连接器和管端,重新连接管端端与Luer Lock耦合器,并用附带的过滤器关闭蓄水池。

  4. 通过将泵连接到流体单元,电源和计算机并调整ibidi ®泵系统软件中的设置来设置泵:
    1. 将泵连接到笔记本电脑并连接所有管道,如图3所示。请注意,带有吸收珠的烧瓶必须放置在泵和流体单元之间,以在潮湿的培养箱中孵育期间除去管中产生的水分。
    2. 启动ibidi ®泵的软件。
    3. 对于2ml / min流量的单向流量,请选择图4中给出的参数。


      图3.计算机和泵系统的设置。 (A)中显示的系统部分保留在工作台上,而流体单元放置在CO 2培养箱内。泵,吸收器和计算机之间的连接在B中显示:泵(1)连接到电源(2)和计算机(3)。用于施加压力的管道安装在泵(4)上,并连接到吸收器瓶(5)。从吸收器瓶中,长管(6)将与孵化器中的流体单元连接,以对储存器中的介质施加压力。该泵包含几个通道用于连接流体单元(C,由箭头指示)。一个泵可以控制多达四个流体单元。在软件中设置实验时选择正确的连接(见图4)

      图4. ibidi ®泵控制软件设置,流速为2 ml / min。 :一种。打开软件并打开调度程序向导概述,方法是单击A1中的按钮。将打开一个新窗口。在我们的应用(Polke等人,,2017)中,使用了A2中所示的参数:流量2,00ml / min;建议压力13,87毫巴;连续阀'2';连续循环时间45,00秒;摆动阀:'未设定';振荡周期时间:'未设定';时间:86,400秒幻灯片选择:μ-slide VI 0.4 ;灌注套选择:15厘米,ID为0.8毫米(蓝色);粘度0,01。完成参数设置后,按A3所示选择"创建计划"。参数将出现在泵控制中;在这一点上,软件和泵可以使用。要将流体单元/灌注连接到泵(按照本协议的步骤7),选择合适的泵通道(B1,也请参见图3),施加压力(B2)并启动流量(B3)。

  5. 将白色念珠菌胚芽管和种子收集在ibidiμ-载玻片上
    1. 为了收集胚芽管,将室温下以4,000×g / g的步骤1j制备的培养基在50ml Falcon管中离心10分钟至20分钟。虽然收集细菌管可能很棘手,有时需要长时间的离心步骤,但是在手中,细菌诱导后30分钟的细菌管可以通过在室温下以4,000 xg离心10分钟来成功收集。
    2. 小心地移除上清液,使用移液管吸出(因为细胞沉淀可能会"蓬松",当倾倒上清液时,它很容易损失)。将细胞重悬于10ml新鲜(预温至37℃)RPMI1640中,以获得5×10 5细胞/ ml的最终细胞密度。对于我们的实验,细胞密度不是关键的,因此我们假设在去除上清液期间没有细胞丢失;然而,对于其他应用,可能需要在最终再悬浮之前重新计数细胞以获得更准确的细胞密度。
    3. 仔细地旋转细胞悬浮液,并通过向每个待使用的通道加入30μl(例如<! - SIPO - >),在ibidiμ-载体VI 0.4 上种白念珠菌细胞, / em>,一个用于流动状态,一个用于静态;参见图5)。通道中的最终细胞数(生长区域的0.6cm 2 / s)为1.5×10 4个细胞/ ml。


      图5.细胞接种到ibidiμ-载玻片VI 0.4 小心地将细胞悬浮液移至无菌条件(A)下μ-载玻片的小储存器中。 30μl就足以填充细胞通道,如果您希望也可以应用更多的培养基。关闭盖子(B),并将μ-载玻片置于培养箱(37℃5%CO 2加湿室)中20分钟以使细胞粘附。

    4. 用包含的盖子关闭通道,并在37℃和5%CO 2下孵育20分钟,以使细菌管粘附到塑料表面。在流动期间,所有非粘附细胞将被洗去并积聚在储存器中的培养基中
  6. 洗涤细胞并将ibidiμ-载玻片连接到灌注组
    1. 粘附后,小心地从通道中取出培养基,通过加入150μl预热的RPMI1640培养基洗涤细胞一次并再次吸出。这将消除大多数非贴壁细胞。避免剧烈的移液,因为这可能会分裂细胞。
    2. 对于在静态条件下生长的细胞,应用120μl预热的RPMI1640培养基,并用盖子封闭通道。
    3. 对于在流动条件下生长的细胞,将约200μl预热的RPMI1640培养基加入适当的通道(通道应完全充满流体[根据图6A])。
    4. 从培养箱中取出流体装置,包括灌注装置,并将其放在干净的工作台上。打开Luer Lock Coupler,并将Luer连接快速连接到通道每端的小型水箱(参见图6)。避免介质溢出管道。当使用夹子临时关闭管道时,这很容易完成,如图6所示。现在准备ibidiμ-载玻片以进行孵育,并且可以固定在流体单元上,如图6D所示。


      图6.将ibidiμ-slide VI 0.4 连接到灌注组(在无菌条件下)。 使用标准夹子关闭管子,以避免连接过程中管道的中度泄漏。卸下Luer Lock耦合器,并将管的每一端连接到其中一个小水箱(A)。确保通道完全充满流体,以避免将气泡引入系统。覆盖在静态条件下生长的细胞(B)。将μ滑块放在流体单元(C)上的指定空间上,并用胶带固定滑块,以避免运输过程中的运动或滑动(D)。然后细胞分别在流动(E1)或静态条件(E2)下生长
    5. 将灌注组的管与阀组正确布置连接:在运行/流动期间,来自一个储存器的介质通过管和μ滑动通道泵入另一个储存器,反之亦然 。为了允许通道中的单向流动,流体单元包含在一个或另一个方向上泵送时切换的两个不同的阀。在制造商的网站上给出了原理的一个很好的描述( https://ibidi.com/perfusion-system/112-ibidi-pump-system.html )和(Wilson和Hube,2010)。为了允许阀的正确切换,管必须以交叉顺序固定(见图7A和8)。


      图7.流量应用将管道与阀门(A)连接。确保按照正确的顺序放置它们,以允许在μ通道中单向流动。按照图中所示的交叉顺序设置。箭头表示有助于以正确顺序放置分支管的标记。将流体单元连接到泵上,并将流体单元放入培养箱(B)中。应用流量并检查储层之间的正确泵送。孵育6 h(或所需时间)。


      图8.管道连接和流动原理。 :一种。设置管道系统。注意,从两个来自每个储层的管中,每个管连接。 B.阀门系统原理及流向。在泵内,来自在泵之后联接的两个储存器的管被放置在相同的阀中。

  7. 将包括灌注装置和ibidiμ-slide在内的Fluidic装置放在培养箱中(图7B)。
  8. 通过软件启动泵来应用流量。检查流量是否正确。应该发生储层之间不平衡的介质流动,因为这将导致空气在这段时间内的吸入。通过检查两个储层中的介质量可以很容易地检测出不平衡的介质流量:这导致两个储层中的介质分布不均匀,这不会降低,而是在开始流动后增加。如果流动顺利运行,请关闭孵化器门。在这样做的同时,避免管道和电缆的瘫痪,因为扭结会阻碍介质流动。在静态/流动条件下孵育酵母所需的时间。
  9. 为了避免积聚在储存介质中的群体感应分子和废物在实验过程中每小时将培养器中的流体单元从泵中和计算机中分离出来。在无菌条件下快速开放水库,移除介质中〜10 ml(总储存量约为11.3 ml)。不要吸入太多的介质,以避免管中气泡形成。立即用新鲜的RPMI1640培养基(10ml,预温至37℃,用5%CO 2平衡)来代替去除的液体。
    注意:在中期交换期间,请勿从流体单元中取出ibidiμ-slide!
  10. 孵化结束时,从培养箱中取出流体装置,将μ-载玻片从灌注套管中取出,并从每个通道中取出培养基。
  11. 仔细冲洗含有细胞的每个通道三次,每次150μl1x PBS。然后,加入120μlHistofix(或4%多聚甲醛)将细胞固定在室温下1 h。用120μl1x PBS替代Histofix进行显微镜检查

数据分析

使用ZEISS AxioVert倒置显微镜和ZEN软件进行显微镜评估。

  1. 启动软件。
  2. 启动显微镜,将ibidi室放在微型载玻片上并进行调整。
  3. 确保在显微镜软件中选择合适的放大倍率。您可以在设置"显微镜组件"中找到各自的选择(用于物镜,变焦和相机适配器)。这对于软件从像素计算出正确的μm长度很重要。
  4. 拍照确保在整个通道中选择几个随机斑点,以在显微镜下定量细丝长度。在我们的研究(Polke等人,2017年)中,我们分析了每个重复的每个通道中的70个随机细胞的长度(总共五个重复)。
  5. 对于灯丝长度的分析,将"缩放"设置为"理论",并在"缩放"设置中选择所需的单位(μm通常是合适的)。
  6. 要测量灯丝长度,请选择"图形"文件夹中的工具"活动曲线"。这允许精确的长丝长度确定。这是很重要的,因为菌丝并不总是直线增长,但可能是弯曲的(见图9),这将在直线上测量时伪造长丝长度确定。对于长丝长度测量,不用母本酵母,但包括尚未分离的芽殖酵母(假菌丝,见图9)。


    图9.长度测量 C。白细胞eed1 使用ZEN软件(ZEISS)在流动(左)或静态(右)条件下生长的Δ细胞。 使用ZEN软件(Blue edition,ZEISS)的活性曲线工具测量每根细丝的长度。长度的测量部分用红线表示。

  7. 测量在静态和流动条件下生长的长丝的长度。或者,可以通过相对于总细胞数计数细丝数来确定细丝诱导。
  8. 在我们的研究中,通过使用GraphPad Prism 5软件的非参数Mann-Whitney测试统计分析了静态或流动条件下的长丝长度(参见图10)。


    图10. C的灯丝长度的定量。在流动或静态条件下生长的白色念珠菌EED1 删除菌株( eed1 Δ) (图片取自[Polke等人,2017])。通过使用GraphPad Prism的非参数Mann-Whitney测试统计分析静态或流动条件下的长丝长度5软件。条中的数字表示在给定生长条件下的三个独立生物实验中,70个细胞的平均长丝长度

笔记

  1. 一些ibidi ®泵系统材料可以通过高压灭菌灭菌并重新用于实验装置。这些包括管道和连接的储存器,但不包括安装的过滤器。为了灭菌,将管道和储存器放置在热稳定的高压灭菌袋(例如,处理袋)中,使用蒸汽指示剂带封闭袋子,并在121℃的标准实验室高压釜中灭菌20分钟。对于过滤器的灭菌,用70%乙醇擦拭过滤器数次,然后在UV光下在UV交联剂(120mJ / cm 2)或相当的UV装置中灭菌15分钟。但是,我们建议不要重复使用管道和水库三次以上。
  2. 在我们出版物中用于实验的流动测定系统中,只有一个流体单元(ibidi)可用并安装在单元上产生单向流。然而,一个ibidiμ-幻灯片VI 0.4 具有六个通道用于细胞播种。因此,如果安装了多个流体单元,则可以在流动条件下生长多于一个样品。如果适用于您的科学问题,请始终将静态样本作为幻灯片上的控件。
  3. 当我们使用ibidi ®泵系统产生单向流来洗涤细胞培养上清液中的积聚QSM时,该系统也可用于模拟生物膜表面上的流动,以模拟血液流程(Wilson和Hube,2010)和更多的应用程序。在制造商的主页上提供了ibidi ®泵系统原理的详细转录,单向流量保证和更多的应用,在制造商的主页上( http://ibidi.com/ )。
  4. 其他制造商的其他流量系统当然可以用于这种类型的实验。选择这里描述的实验系统的重要方面是容易从系统中移除哪种介质,以及相对于通道体积的总介质体积,因为这决定了稀释率。例如,对于ibidi ®泵系统,也有更大的油藏组(50毫升)。

食谱

  1. 30%甘油溶液(100ml)
    30ml甘油,ROTIPURAN ,无水
    加入70毫升去离子水 高压灭菌器
  2. 20%D(+) - 葡萄糖溶液(500 ml) 100克D(+) - 葡萄糖溶液,无水
    加入去离子水至500 ml
    高压灭菌器
  3. YPD肉汤(500毫升)
    2%(w / v)(10g)Bacto TM蛋白胨
    1%(w / v)(5g)酵母提取物,微粒化的
    加入去离子水至450毫升,高压釜
    加入50ml无菌20%D(+) - 葡萄糖溶液(终浓度2%)
  4. YPD琼脂培养基(500ml) 2%(w / v)(10g)Bacto TM蛋白胨
    1%(w / v)(5g)酵母提取物,微粒化的
    2%(w / v)(10g)D(+) - 葡萄糖
    2%(w / v)琼脂,神户我
    高压灭菌器
  5. 10倍PBS
    1.5 M NaCl
    100mM Na 2 HPO 4•2H 2 O
    12mM KH PO 4
    将pH调节至7.4
    高压灭菌器
  6. 1x PBS
    100毫升10倍PBS,加入900毫升无菌水
  7. 70%乙醇(1升)
    混合700 ml乙醇(变性,≥99.8%)和300 ml去离子水

致谢

这项工作得到了德国电力公司Vol。e.V的财务支持。 (MP)和德意志民主共和国(DFG JA1960 / 1-1至IJ)。该方案适用于Wilson和Hube(2010)。感谢Duncan Wilson博士和Sascha Brunke博士有关ibidi ®泵系统设置的有用建议。

参考

  1. Hornby,JM,Jensen,EC,Lisec,AD,Tasto,JJ,Jahnke,B.,Shoemaker,R.,Dussault,P.and Nickerson,KW(2001)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/11425711"target ="_ blank">二倍体真菌中的法定感测白色念珠菌由法呢醇介导。 Appl Environ Microbiol 67(7):2982-2992。
  2. Lindsay,AK,Deveau,A.,Piispanen,AE和Hogan,DA(2012)。&nbsp; 对于白色念珠菌中的菌丝酵母转化的法尼醇和环AMP信号传导效应真核细胞 11(10) :1219-1225。
  3. Polke,M.,Sprenger,M.,Scherlach,K.,Alban-Proano,MC,Martin,R.,Hertweck,C.,Hube,B.and Jacobsen,ID(2017)。白色念珠菌中的菌丝维持和群体感应之间的功能链接Mol Microbiol 103(4):595-617。
  4. Wilson D.和Hube,B。(2010)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20023069"target ="_ blank" > Hgc1介导动物白色念珠菌循环期间的内皮粘连事件。真核细胞 9(2):278-287。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Polke, M. and Jacobsen, I. (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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