MAMP-triggered Medium Alkalinization of Plant Cell Cultures

Materials and Reagents

1. 12-well cell culture plate (Greiner Bio-One, CELLSTAR^®, catalog number: 665180 )
2. Serological pipette without tip 10 ml (SARSTEDT, catalog number: 86.1688.010 )
3. P200 pipette tips
4. 20 ml syringe (VWR, catalog number: 613-2046 )
5. 0.22 µm syringe filter (GE Healthcare, Whatman^TM Uniflo, catalog number: 9915-2502 )
6. Hexa-N-acetylchitohexaose (two manufacturers are recommended: Santa Cruz Biotechnology, catalog number: sc-222018 ; Isosep, catalog number: 56/11-0010 ). Store at -20 °C
   Note: Chitoheptaose (NAG7) and chitooctaose (NAG8) or even chitin (Sigma, catalog number: C7170) may be used instead of chitohexaose.
7. Flagellin 22 peptide (flg22: QRLSTGSRINSAKDDAAGLQIA) (Genscript, catalog number: RP19986 ). Store at -20 °C
8. Gamborg’s vitamin Solution 1,000x (sterile) (Sigma-Aldrich, catalog number: G1019-50ML ). Store at 4 °C
   Note: Before using, inspect for the presence of precipitates. If present, warm the solution up to 42 °C and vortex until all precipitates dissolve.
9. Murashige and Skoog Basal salt mixture (PhytoTechnology Laboratories, catalog number: M524 ). Store at 4 °C
10. Sucrose (Sigma-Aldrich, catalog number: S5391 )
11. Myo-inositol (Sigma-Aldrich, catalog number: I7508 )
12. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 ). Store at 4 °C
13. KH₂PO₄ (Sigma-Aldrich, catalog number: P5655 )
14. Tryptone (Oxoid, catalog number: LP0042 )
15. 2,4-Dichlorophenoxyacetic acid (Sigma-Aldrich, catalog number: D7299 )
16. 2-(N-Morpholino)ethanosulfonic acid (MES) hydrate (Sigma-Aldrich, catalog number: M2933 ) 
17. Kinetin (Sigma-Aldrich, catalog number: K0753 )
18. Chitohexamers (NAG6) solution (see Recipes)
19. Flg22 solution (see Recipes)
20. Kinetin stock solution (1,000x) (see Recipes)
21. 2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution (1,000x) (see Recipes)
22. KH₂PO₄ stock solution (see Recipes)
23. Thiamine/myo-inositol stock solution (see Recipes)
24. Tryptone stock solution (10%) (see Recipes)
25. MES/Phosphate-buffered culture medium (see Recipes)
26. Subculturing medium (see Recipes)
    

Equipment

1. 125 ml Erlenmeyer flasks (Pyrex^TM, catalog number: 15685767 )
2. 250 ml Erlenmeyer flasks (Pyrex^TM, catalog number: 11902619 )
3. P200 adjustable pipette
4. Flow hood (Thermo Scientific, model: 1300 Series Class II , catalog number: 1323TS )
5. Autoclave (VWR, catalog number: 481-0666 )
6. pH meter (Mettler Toledo, Seven Compact S220 Basic, catalog number: 30019028 ). Alternative models may be used as long as the pH resolution is at least 0.001 
7. pH sensor InLab Micro for small volumes (Mettler Toledo, catalog number: 51343160 )
8. Orbital shaker (LaboTech, model: RS150 )
   

Software

1. Microsoft Excel
2. EasyDirect^TM pH Software (Mettler Toledo, 30323214)
   

Procedure

1. Cell suspension preparation and maintenance
   The protocol described here uses Nicotiana tabacum cultivar Bright Yellow 2 (BY-2) cell suspensions, which are established from in vitro-induced calli (Figure 1A). The BY-2 cell line was produced from tobacco seedlings by Kato et al. in 1972 (Kato et al.,1972) and has since been propagated and shared among plant scientists (Nagata et al., 1992). BY-2 cells are currently used as models in a number of experimental systems and are available from various research groups.
   
   1. To generate a suspension of plant cells, transfer approximately 1 ml of calli to a sterile 125 ml Erlenmeyer flask containing 20 ml of MES/Phosphate-buffered culture medium (Recipe 8) using a sterile spatula or inoculation loop (in the flow hood). Be gentle to avoid unnecessary mechanical stress to the calli. In order to prevent contamination, work carefully and orderly at the flow hood. Incubate at 27 °C in the dark and under gentle agitation (100-120 rpm) in an orbital shaker. The calli will release small cell aggregates that will eventually result in a cell suspension.
   2. After 10-14 days, transfer 5 ml of the resulting cell suspension using a 10 ml tipless serological pipette to a sterile 250 ml Erlenmeyer flask containing 45 ml of subculturing medium (Recipe 9). Incubate at 27 °C in the dark and under gentle agitation (100-120 rpm) in an orbital shaker.
   3. After 7 days, repeat the subculturing as described in Step A2. At this point, the newly established cell suspension should be similar to the one shown in Figures 1B-1C. Inspect an aliquot of the suspension under the microscope to ensure that it is free from contaminants.
   4. For regular maintenance of BY-2 cell suspensions, subculture the cells weekly as described in Step A2. It is advisable to routinely keep two or three independent flasks of cell suspensions to prevent culture loss due to contamination.
      
      
      Figure 1. Nicotiana tabacum cv. Bright Yellow 2 (BY-2) cells growing as calli and cell suspensions. A. Calli growing on solid MES/Phosphate-buffered culture medium. B. Cell suspension just after subculturing. C. A 7-day-old cell suspension. Note the pale-yellow color indicating saturation.
      
      
2. MAMP treatment and measurement of medium alkalinization
   Once established cell suspensions are available (as described in the previous section), MAMP-triggered medium alkalinization can be accessed. Aliquots of cell suspension are individually treated with MAMPs and probed for response (Figure 2).
   
   
   Figure 2. Experimental setup for medium alkalinization measurement. A 12-well microplate containing 2.5 ml of cell suspensions in each well is kept under gentle agitation on an orbital shaker. The pH sensor is inserted so that the probe is completely submerged and does not touch the walls.
   
   
   1. Carefully transfer a 3- to 5-day-old cell suspension to a 12-well plate (2.5 ml per well) using a 10 ml tipless serological pipette. The cell cultures to be used should be similar to the ones shown in Figure 1C, with a pale-yellow coloration. Dark yellow or brownish colored suspensions are typically stressed and should not be used.
      Note: One key factor that affects the medium alkalinization assay is the age of the cell suspension. In our experiments with BY-2 cells, we used 3- to 5-day-old suspensions. However, the optimum age may vary from lab to lab and therefore needs to be determined experimentally.
   2. Incubate the plate for at least 2 h and up to 4 h at room temperature and under gentle agitation in an orbital shaker (typically 100-200 rpm; the speed should be just enough to keep the cells in suspension). This step allows the cells to recover from stresses caused by the transference to the plate.
   3. Before starting the measurements, adjust the pH meter settings appropriately: measurement resolution of 0.001, endpoint format set as manual and timed interval readings of 3 s. Calibrate the sensor according to the manufacturer’s recommendations using calibration standard solutions.
   4. Open the EasyDirect^TM pH Software on a computer connected to the pH meter and set the export option to Excel. An Excel sheet will automatically be opened.
   5. Keeping the plate under agitation on the orbital shaker (100-200 rpm), insert the sensor in one well, carefully adjusting the position to ensure that the sensor is sufficiently inserted (colored tip submerged) and is not touching the walls or the bottom of the well.
   6. Press the Start button on the pH meter. The instrument will start recording the pH values in the Excel sheet.
   7. Once the measurements become stable (same value for at least 12 s), add 25 μl of 1 µM NAG6 (Recipe 1) (final concentration: 10 nM) or 25 μl of 10 µM flg22 (Recipe 2) (final concentration: 100 nM) to the well. To avoid artefactual changes in pH, equilibrate the elicitors to room temperature for 10 min before adding to the cell suspension. Make sure that each plate includes at least one well treated with elicitor solvent (H₂O) as non-elicited control.
   8. Record the pH values for 10 min for NAG6 and 60 min for flg22.
   9. After the measurement is complete, remove the sensor from the well, wash thoroughly with ultrapure water and dry using tissue paper.
   10. Repeat Steps B5-B9 until all samples included in the experiment have been tested.
       

Data analysis

The EasyDirect^TM pH Software features an export tool that records pH measurements directly into Excel sheets. Therefore, once all the raw data from an experiment is collected, the variation in pH (ΔpH) over time for each tested condition can be calculated by subtracting the pH value measured at time 0 (i.e., the pH of the medium before MAMP treatment) from the pH at each subsequent time point (i.e., the pH of the medium after MAMP treatment). Line plots depicting the variation in pH over time (Figure 3A) or bar plots showing the maximum ΔpH value (Figure 3B) for each treatment may be plotted to visualize the results. Standard deviation should be presented to indicate variation among replicates. It is desirable to have at least 3 biological replicates for each condition tested.


Figure 3. NAG6-induced medium alkalinization of tobacco BY-2 cell suspensions. The pH of cell suspensions treated with 10 nM NAG6 or ultrapure water (mock) was recorded for 10 min after the treatment. A. Variation in pH (ΔpH) over time (in seconds) shown in a line plot. B. Maximum variation in pH plotted as a bar graph. Shades in the line graph and error bars in the bar plot indicate standard deviation of three biological replicates.

Notes

1. As an alternative to the tipless serological pipette, it is possible to cut the end of a regular serological pipette or even from a P5000 tip and use it for handling of the cell suspensions.

Recipes

1. Chitohexamers (NAG6) solution
   Dissolve in sterile ultrapure water to 1 µM (1.24 µg/ml)
   Store at -20 °C
2. Flg22 solution
   Dissolve in sterile ultrapure water to 10 µM (22.7 µg/ml)
   Store at -20 °C
3. Kinetin stock solution (1,000x)
   
   1. Dissolve 0.1 mg/ml kinetin in 1/10 volume (100 µl per ml) of 1 M NaOH aqueous solution
      
   2. Once the powder is dissolved, complete the final volume with ultrapure water
      
   3. Filter sterilize and store at -20 °C
4. 2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution (1,000x)
   Dissolve 1 mg/ml 2,4-D in absolute ethanol
   Store at -20 °C
5. KH₂PO₄ stock solution
   
   1. Dissolve 60 mg/ml KH₂PO₄ in deionized water
      
   2. Prepare 10 ml aliquots and store at -20 °C
6. Thiamine/myo-inositol stock solution
   
   1. Dissolve 0.1 mg/ml thiamine hydrochloride and 10 mg/ml myo-inositol in deionized water
      
   2. Prepare 3 ml aliquots and store at -20 °C
7. Tryptone stock solution (10%)
   
   1. Dissolve 10 g/L tryptone in deionized water
      
   2. Sterilize by autoclavation or filtration through a 0.22 µm syringe filter
      
   3. Store at room temperature
8. MES/Phosphate-buffered culture medium
   Composition:
   0.5 g/L 2-(N-Morpholino)ethanosulfonic acid (MES) hydrate
   30 g/L sucrose
   1x Murashige and Skoog basal salts
   100 mg/L myo-inositol
   1 mg/L Thiamine hydrochloride (vitamin B1)
   0.18 g/L KH₂PO₄
   0.22 mg/L (0.1 µM) 2,4-Dichlorophenoxyacetic acid
   1% tryptone
   pH 5.7
   
   Preparation (1 L):
   
   1. Dissolve 4.3 g of Murashige and Skoog basal salt mixture, 30 g sucrose and 0.5 g MES hydrate in 900 ml of deionized water
      
   2. Adjust the pH to 5.7 using a 1 M KOH aqueous solution
      
   3. Add 3 ml of KH₂PO₄ stock solution, 10 ml of Thiamine/myo-inositol stock solution and 220 µl of 2,4-D stock solution
      
   4. Complete the final volume to 1 L with deionized water
      
   5. Transfer 18 ml aliquots to 125 ml Erlenmeyer flasks
      
   6. Autoclave at 121 °C and 15 psi for 20 min
      
   7. After equilibration at room temperature, add 2 ml of tryptone stock solution. The medium is ready to be used

9. Subculturing medium
   Composition:
   1x Murashige and Skoog basal salts
   30 g/L sucrose
   1x Gamborg’s vitamins
   1 mg/L (4.5 µM) 2,4-Dichlorophenoxyacetic acid
   0.1 mg/L (0.46 µM) kinetin
   pH 5.7
   
   Preparation (1 L):
   
   1. Dissolve 4.3 g of Murashige and Skoog basal salt mixture and 30 g sucrose in 900 ml of deionized water
      
   2. Adjust the pH to 5.7 using a 1 M KOH aqueous solution
      
   3. Complete the final volume with deionized water
      
   4. Transfer 45 ml aliquots to 250 ml Erlenmeyer flasks
      
   5. Autoclave at 121 °C and 15 psi for 20 min
      
   6. After equilibration at room temperature, transfer the autoclaved flasks to a flow hood and add 45 µl of 1,000x Gamborg’s vitamin solution (sterile), 45 µl of 2,4-Dichlorophenoxyacetic acid stock solution and 45 µl of kinetin stock solution. The medium is ready to be used

Acknowledgments

This protocol describes in detail the procedure to access MAMP-triggered medium alkalinization of cell suspensions used in the research report by Fiorin et al. (2018). This research was supported by the São Paulo Research Foundation (FAPESP) through fellowships 09/51018-1, 09/50119-9, 11/23315-1, 13/09878-9 and 14/06181-0.

Competing interests

The authors declare no conflict of interest.

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