Plant material for the analysis of the root metabolome was collected in the same experiment as that earlier used for pea leaf metabolome analysis [5 (link)]. The samples of plant material (0.2–0.3 g) were ground as described by Puzanskiy et al. [55 (link)] and subjected to a single-stage extraction with 2 mL methanol: chloroform: water (5:2:1) mixture. Tissue debris was removed by centrifugation at 12,000× g for 10 min at −5 °C. The supernatant was collected and evaporated in a vacuum evaporator (Eppendorf, Germany). The dried material was dissolved in pyridine with the internal tricosane standard (nC23, tricosane). The samples were then supplied with the sylilating agent BSTFA:TMCS 99:1 (Sigma-Aldrich) and derivatizated at 90 °C for 20 min.
Vacuum evaporator
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Sourced in Germany
The Eppendorf Vacuum Evaporator is a laboratory equipment used to concentrate liquid samples by removing solvents through evaporation under reduced pressure. It provides a controlled environment to efficiently evaporate a variety of solvents, leaving behind the desired non-volatile components.
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3 protocols using vacuum evaporator
1
Root Metabolome Analysis Protocol
2
Metabolite Extraction for NMR Analysis
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Extraction of aqueous metabolites
from the cells and tissue samples for NMR experiments was carried
out as described earlier.30 (link) MDA-MB-231
cells were cultured and treated with indicated concentrations of DIM
for 24 h. After treatment, cells were scraped off from the flask,
collected into a tube, and washed with cold 1×PBS to remove media
traces. Further, 600 μL of chilled methanol was added to the
cells and mixed using a roto-spin rotary mixer overnight at 4 °C.
Later, 600 μL of distilled water and chloroform were added to
the cell suspension and then vortexed for 30 s and centrifuged at
150×g for 5 min. The upper aqueous fraction
was collected into a new tube and dried by a vacuum evaporator (Eppendorf,
Hamburg, Germany). Before analysis, the extracts were reconstituted
in 55 μL of PBS prepared in D2O (100 mM concentration)
and 495 μL of D2O.
from the cells and tissue samples for NMR experiments was carried
out as described earlier.30 (link) MDA-MB-231
cells were cultured and treated with indicated concentrations of DIM
for 24 h. After treatment, cells were scraped off from the flask,
collected into a tube, and washed with cold 1×PBS to remove media
traces. Further, 600 μL of chilled methanol was added to the
cells and mixed using a roto-spin rotary mixer overnight at 4 °C.
Later, 600 μL of distilled water and chloroform were added to
the cell suspension and then vortexed for 30 s and centrifuged at
150×g for 5 min. The upper aqueous fraction
was collected into a new tube and dried by a vacuum evaporator (Eppendorf,
Hamburg, Germany). Before analysis, the extracts were reconstituted
in 55 μL of PBS prepared in D2O (100 mM concentration)
and 495 μL of D2O.
3
Extraction of Plant Compounds using ASE
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The plants were separated into leaves and stems, dried at room temperature and ground in a Wiley mill. The extraction procedure was chosen on the basis of similar conditions previously reported (Ju and Howard, 2003 (link); Bergeron et al., 2005 (link)). Extractions were conducted using a Dionex ASE 100 system (Oakville, ON, Canada) with stainless steel vessels (66 mL) using 0.5 g of dry ground plant, and ~60 mL EtOH at 60°C and 1500 psi for 15 min. The extracts were dried using a vacuum evaporator (Eppendorf, Hauppauge, NY, USA).
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