6. To confirm the viability of the guard cells, a few samples were
not frozen, but stained with 2.5 mM fluorescein diacetate
(FDA) at room temperature for 5 min.
7. Observe under a fluorescence microscope using a filter cube
that allows an excitation wavelength of 460 nm, and an emission wavelength of 525 nm. Take images under both bright
field and fluorescence excitation.
8. To obtain 100 mg of dried guard cells for each sample, move
15 mL centrifuge tubes with guard cell samples to a 3.8-L
Labconco freeze dryer and dry for 2–3 days.
9. Weigh freeze-dried samples to obtain 100 mg for each replicate
for metabolite extraction. The 100 mg dry weight material is
adequate for at least eight replicate injections under both positive and negative modes of mass spectrometry run.
3.3 Metabolite
Extraction (See Note 3)
1. To each lyophilized guard cell sample, 3 mL methanol, 2 mL
water, and 6 mL chloroform were added in this order and
vortexed at 200 rpm for 1 h at 4
C.
2. After centrifugation at 500 g for 10 min, all the solution was
transferred to a new glass vial, and 2 mL chloroform/methanol
(2:1) with 0.01% BHT was added to the pellet and vortexed for
30 min at 4
C.
3. Repeat the previous extraction procedure twice. All the extracts
were combined and centrifuged at 10,000 g for 10 min.
4. Use a pipette to carefully draw off the upper aqueous phase and
transfer it to a 2 mL microcentrifuge tube.
5. Lyophilize the upper aqueous phase with a Speed Vac and then
store it at À80
C.
6. Resuspend dried metabolite samples in sterile water with 0.1%
formic acid and make a pool sample by mixing equal volumes of
all metabolite samples to prepare for mass spectrometry analysis. Please note that the bottom organic phase can be blown dry
by nitrogen gas, and solubilized in 100% isopropanol for lipid
profiling (see Note 3).
3.4 Mass
Spectrometry Using
Acquire X (See Note 4)
1. Using an untargeted metabolomic method, run the aqueous
phase metabolite samples from Subheading 3.3, step 6 on the
high-resolution Orbitrap Fusion Tribrid mass spectrometer
connected with Vanquish™ UHPLC liquid chromatography.
2. Attach the Accucore C18 (100 Â 2.1) column. Set column
chamber temperature to 55
C.
3. Set pump flow rate to 0.45 mL/min. The LC gradient is set to
0 min: 1% of solvent B (i.e., 99% of solvent A), 5 min: 1% of B,
6 min: 40% of B, 7.5 min: 98% of B, 8.5 min: 98% of B, 9 min:
0.1% of B, 10 min stop run.
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Lisa David et al.
not frozen, but stained with 2.5 mM fluorescein diacetate
(FDA) at room temperature for 5 min.
7. Observe under a fluorescence microscope using a filter cube
that allows an excitation wavelength of 460 nm, and an emission wavelength of 525 nm. Take images under both bright
field and fluorescence excitation.
8. To obtain 100 mg of dried guard cells for each sample, move
15 mL centrifuge tubes with guard cell samples to a 3.8-L
Labconco freeze dryer and dry for 2–3 days.
9. Weigh freeze-dried samples to obtain 100 mg for each replicate
for metabolite extraction. The 100 mg dry weight material is
adequate for at least eight replicate injections under both positive and negative modes of mass spectrometry run.
3.3 Metabolite
Extraction (See Note 3)
1. To each lyophilized guard cell sample, 3 mL methanol, 2 mL
water, and 6 mL chloroform were added in this order and
vortexed at 200 rpm for 1 h at 4
C.
2. After centrifugation at 500 g for 10 min, all the solution was
transferred to a new glass vial, and 2 mL chloroform/methanol
(2:1) with 0.01% BHT was added to the pellet and vortexed for
30 min at 4
C.
3. Repeat the previous extraction procedure twice. All the extracts
were combined and centrifuged at 10,000 g for 10 min.
4. Use a pipette to carefully draw off the upper aqueous phase and
transfer it to a 2 mL microcentrifuge tube.
5. Lyophilize the upper aqueous phase with a Speed Vac and then
store it at À80
C.
6. Resuspend dried metabolite samples in sterile water with 0.1%
formic acid and make a pool sample by mixing equal volumes of
all metabolite samples to prepare for mass spectrometry analysis. Please note that the bottom organic phase can be blown dry
by nitrogen gas, and solubilized in 100% isopropanol for lipid
profiling (see Note 3).
3.4 Mass
Spectrometry Using
Acquire X (See Note 4)
1. Using an untargeted metabolomic method, run the aqueous
phase metabolite samples from Subheading 3.3, step 6 on the
high-resolution Orbitrap Fusion Tribrid mass spectrometer
connected with Vanquish™ UHPLC liquid chromatography.
2. Attach the Accucore C18 (100 Â 2.1) column. Set column
chamber temperature to 55
C.
3. Set pump flow rate to 0.45 mL/min. The LC gradient is set to
0 min: 1% of solvent B (i.e., 99% of solvent A), 5 min: 1% of B,
6 min: 40% of B, 7.5 min: 98% of B, 8.5 min: 98% of B, 9 min:
0.1% of B, 10 min stop run.
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Lisa David et al.