Measurement of Extracellular Ca²⁺ Influx and Intracellular H⁺ Efflux in Response to Glycerol and PEG6000 Treatments

Materials and Reagents

1. Transformed Pichia pastoris cells (Invitrogen, catalog number: V200-20 )
   
2. Poly-L-lysine solution (0.1% w/v aqueous solution) (Sigma-Aldrich, catalog number: P4707 )
   
3. Polyethylene glycol 6000 (PEG6000) (Merck KGaA, catalog number: 807491 )
   
4. Glycerol (Sinopharm Chemical Reagent, catalog number: 10010692 )
   
5. Yeast extract (Oxoid, catalog number: LP0021 )
   
6. Peptone (Oxoid, catalog number: LP0037 )
   
7. D-glucose (Sinopharm Chemical Reagent, catalog number: 10010592 )
   
8. MES
   
9. Standard medium buffer (pH 6.0) (see Recipes)
   
10. Yeast extract peptone dextrose (YPD) medium (see Recipes)
    
11. Calibration medium buffer (pH 7.0) (see Recipes)
    
12. Calibration medium buffer (pH 5.0) (see Recipes)
    

Equipment

1. Non-invasive Micro-test System (YoungerUSA, model: NMT100 series )
   
2. Shaking incubator (Shanghai Anting Scientific Instrument Factory, model: HZQ-F160 )
   
3. Centrifuge (Thermo Fisher Scientific, model: Fresco 21 )
   
4. Microplate Reader Spectra (Molecular Devices, model: SpectraMax 190 )
   
5. Glass coverslide (20 mm x 20 mm)
   
6. Petri dish (35 mm in diameter)
   
7. Micropipettor (Eppendorf, 100-1,000 μl and 10-100 μl)
   

Software

1. JCal V3.2.1 (a free MS Excel spreadsheet, available at http://www.youngerusa.com or http://www.ifluxes.com)
   
2. ASET 2.0 software (available at http://www.youngerusa.com)
   
3. iFluxes 1.0 software (available at http://www.youngerusa.com)
   

Procedure

1. The transformed cells are incubated in 10 ml YPD at 30 °C in a shaking incubator (200 rpm) for 12 h.
   
2. Overnight cultures of different transformed cells are adjusted to an OD_600nm of 0.2. OD_600nm is monitored using microplate reader spectra.
   
3. Fifty microliters of each are taken and added to 10 ml YPD containing a final concentration of 25% PEG6000 or 1 M glycerol or no exogenous osmolytes. The transformed yeast cells are grown to an OD_600nm of 1.0 in YPD containing different exogenous osmolytes at 30 °C in a shaking incubator (200 rpm). OD_600nm is monitored using microplate reader spectra.
   
4. One milliliter of each is taken and pelleted at 2,000 rpm for 5 min at room temperature.
   
5. Nine hundred microliters of supernatant are removed, and the cells are resuspended in the rest of YPD media.
   
6. The coverslips are immersed in the poly-L-lysine solution (0.1% w/v aqueous solution) for 24 h.
   
7. Prior to each flux measurement, the microelectrodes must be calibrated in calibration medium (pH 7.0 and pH 5.0), respectively, following to the same procedure and standards. Only Ca²⁺ electrodes with Nernstian slope > 26 mV/decade and H⁺ electrodes with Nernstian slope > 53 mV/decade are used in the protocol. Data are discarded if the post-test calibrations fail.
   
8. Ten microliters of transformed cells are loaded on the coverslip for 5 min, washed off with standard medium to ensure a monolayer of attached cells and incubated in the standard medium for 5 min at room temperature.
   
9. Microelectrodes are positioned 10 μm above the attached cell population consisting of 15 cells with equal size. Micro-volts differences are measured at two excursion points, one 10 μm above the cell population and the other 20 μm away, at a frequency of 0.05 Hz manipulated by a computer. The kinetics of net Ca²⁺ and H⁺ fluxes near each cell population are monitored for 10 min.
   
10. For each sample, four clones are incubated in 10 ml YPD, and the resulting four cell populations are measured (see steps 1-9).
    
11. Micro-volts differences are exported as raw data before they are converted into net Ca²⁺ and H⁺ fluxes by using the JCal V3.2.1. The ion flux assay around each type of transformed cells is replicated independently three times.
    
    
    Figure 1. Schematic diagram of ion flux detection (www.xuyue.net). The microelectrode tip is filled with liquid ion exchanger (LIX). A voltage gradient (dV) is measured by the electrometer between two positions over the travel range dx. A concentration gradient (dc) is calculated based on dV. D_o, ion diffusion constant; J, net ion flux.
    

Recipes

1. Standard medium (aqueous solution) buffer (pH 6.0)
   0.1 mM CaCl₂
   0.1 mM KCl
   0.3 mM MES
   10 mM glucose
   pH is adjusted to 6.0 with HCl
   Stored at 4 °C
   
2. Yeast extract peptone dextrose (YPD) medium (1 L)
   1% yeast extract
   2% peptone
   2% D-glucose (added to the medium after autoclave)
   10 g yeast extract
   20 g peptone are dissolved in 900 ml of water
   The medium is autoclaved for 20 minutes on liquid cycle, cooled to ~55 °C
   Mixed with 100 ml of 20% D-glucose
   The liquid medium is stored at room temperature
   
3. Calibration medium (aqueous solution) buffer (pH 7.0)
   0.01 mM CaCl₂
   0.1 mM KCl
   0.3 mM MES
   10 mM glucose
   pH is adjusted to 7.0 with HCl
   The medium is stored at 4 °C
   
4. Calibration medium (aqueous solution) buffer (pH 5.0)
   0.1 mM CaCl₂
   0.1 mM KCl
   0.3 mM MES
   10 mM glucose
   pH is adjusted to 5.0 with HCl
   The medium is stored at 4 °C
   

Acknowledgments

The protocol was adapted from our previously published paper Li et al. (2013). We wish to thank Younger USA (Xuyue Beijing) NMT Service Center for the technical support. This research was financially supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (Project no. KZCX2-YW-BR-17) and National Natural Science Foundation of China (41371264, 41401281).

References

1. Li, T., Hu, Y. J., Hao, Z. P., Li, H., Wang, Y. S. and Chen, B. D. (2013). First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus Glomus intraradices. New Phytol 197(2): 617-630.
   
2. McLamore, E. S. and Porterfield, D. M. (2011). Non-invasive tools for measuring metabolism and biophysical analyte transport: self-referencing physiological sensing. Chem Soc Rev 40(11): 5308-5320.
   
3. Shabala, L., Ross, T., McMeekin, T. and Shabala, S. (2006). Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment. FEMS Microbiol Rev 30(3): 472-486.
   
4. Shabala, L., Ross, T., Newman, I., McMeekin, T. and Shabala, S. (2001). Measurements of net fluxes and extracellular changes of H⁺, Ca²⁺, K⁺, and NH₄⁺ in Escherichia coli using ion-selective microelectrodes. J Microbiol Methods 46(2): 119-129. 
5. Wang, Q., Zhao, Y., Luo, W., Li, R., He, Q., Fang, X., Michele, R. D., Ast, C., von Wiren, N. and Lin, J. (2013). Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization. Proc Natl Acad Sci U S A 110(32): 13204-13209.
   
6. Xu, R. R., Qi, S. D., Lu, L. T., Chen, C. T., Wu, C. A. and Zheng, C. C. (2011). A DExD/H box RNA helicase is important for K+ deprivation responses and tolerance in Arabidopsis thaliana. FEBS J 278(13): 2296-2306.