Surface-sterilized Arabidopsis seeds were sowed on 0.5 x MS medium .
{"action": "sow", "output": "", "container": ["Surface-sterilized"], "temperature": [""]}
kept for 3 d at 4 °C in the dark
{"action": "keep", "output": "", "temperature": ["4 \u00b0C"]}
The plates were then transferred to the growth chamber at 22 °C with a 16-h light/8-h dark photoperiod .
{"action": "transfer", "output": "", "container": ["the growth chamber"], "temperature": ["22 °C"]}
Pt–GFP Root hairs of 4-5-d-old seedlings were used to monitor intracellular pH. #####%%%%% output "intracellular pH" are not used
{"action": "use", "output": "intracellular pH", "reagent": [""]}
Seedlings were carefully placed on the slide with 2-3 drops of liquid growth medium (the same as the medium seedling were grown on).
{"action": "place", "output": "", "reagent": ["liquid growth medium"]}
Root hair GFP fluorescence was monitored under the confocal microscope (exciters: 405 nm ; emitter: 525 nm); the F405-to-F488 ratio was used as a measure for pH, #####%%%%% output "F488" are used in line16
 {"action": "monitor", "output": "F488", "device": ["confocal microscope"]}
The pH titrations were performed in situ as previously reported Moseyko .

For making the calibration curve, a series of pH buffer solutions (5, 5.5, 6, , 7, ; for preparation, see Recipes) were used, 4-5-d-old seedlings were incubated in the buffer solution . #####%%%%% output "the calibration curve" are used in line24
{"action": "operate", "output": "the calibration curve", "reagent": ["pH buffer solutions", "the buffer solution", "F488"], "device": [""]}
were stabilized for about 10 min to equilibrate the external pH buffer solutions
{"action": "recover", "output": "", "time": ["about 10 min"], "container": ["the external pH buffer solutions"], "temperature": [""], "volume": [""]}
Fluorescence was made as procedure 1 to obtain F405-to-F488 ratio for different pH buffer solution. #####%%%%% output "F405-to-[F488] ratio" are not used
{"action": "make", "output": "F405-to-[F488] ratio", "concentration": [""], "device": ["fluorescence"]}
For each, at least 5 repeats should be made.
{"action": "make", "output": "", "volume": [""]}
After the in situ titration, an equation (R2 near 0.999) will be reached for conversion the fluorescence ratio to the pH value by using the Microsoft Excel. #####%%%%% output "in situ titration" are not used
{"action": "confirm", "output": "in situ titration", "device": ["the Microsoft Excel"], "reagent": ["the calibration curve"]}
pH levels of the root hair obtained in step 1 were then calibrated by the equation.
{"action": "calibrate", "output": ""}