Sample preparation was carried out as described previously (32 (link)). C. carassius cultured at 20 or 30°C were euthanized in ice slush (5 parts ice/1 part water, 0–4°C) for at least 10 min following cessation of gill movement, and left in the ice water for a total of 20 min after cessation of all movement to ensure death by hypoxia following the guidelines of NIH. C. carassius were rinsed with distilled water and then wiped thoroughly with sterilized gauze. Spleens were removed ascetically, where 25 mg of spleens were cut and immensed immediately in 1 mL cold methanol. Then, the samples were sonicated for 5 min at 10W-power setting at Ultrasonic Processor (JY92-IIDN, SCIENTZ), followed by centrifugation at 12,000 × g in 4°C for 10 min. The supernatant was collected and 10 μl 0.1 mg/ml ribitol (Sigma) was added as the internal standard. The supernatant was concentrated in a rotary vacuum centrifuge device (LABCONCO). The dried polar extracts were incubated with 80 μl methoxy amination hydrochloride (20 mg/ml pyridine) for 90 min at 37°C, followed by an addition of 80 μl N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (Sigma) and incubated for another 30 min at 37°C. Finally, the resulted samples were cooled down to room temperature prior to mass spectrometry analysis.
Rotary vacuum centrifuge device
Manufactured by Labconco
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The Rotary vacuum centrifuge device is a laboratory equipment used to separate and concentrate liquid or solid samples under vacuum. It utilizes centrifugal force to separate components based on their density differences. The device provides a controlled environment for sample processing and can be used for various applications in scientific research and analysis.
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6 protocols using rotary vacuum centrifuge device
1
Metabolomic Analysis of Goldfish Spleen
2
Zebrafish Metabolite Extraction for GC-MS
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Zebrafish body fluid was collected as previously described with a few modifications [14 (link)]. In brief, zebrafish were rinsed with distilled water and then wiped thoroughly with sterilized gauze. These animals were cut into five pieces on ice and then weighted. The appropriate volume of saline (100 μL/100 mg) was added according to the weight. After centrifugation at 3000 × g, 4 0C, 100 μL fluid was isolated for the further study of metabolites. Metabolites were extracted with 0.2 mL cold methanol (Sigma) containing 10 μL 0.1 mg/mL ribitol (Sigma) as an analytical internal standard. After centrifugation at 12,000 × g for 10 min, the supernatant was concentrated in a rotary vacuum centrifuge device (LABCONCO). The dried polar extracts were used for GC-MS analysis.
3
Liver Metabolomics in Goldfish
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The sample preparation was carried out as previously described (32 (link), 47 (link)). Individual livers from each fish were surgically removed and quickly rinsed with cold saline buffer to remove blood. Then, cold methanol was added at 800 μl/100 mg. The whole liver was homogenized in methanol, followed by sonication for 5 min at a 10-W power setting. A total of 30 C. carassius accommodated at 26°C were used in this experiment (n = 10 for each group). The liver from individual C. carassius was treated as one biological sample. Samples were then centrifuged to remove unresolved matter at 12,000 × g, 4°C for 10 min. The supernatant was collected, and 10 μl 0.1 mg/ml ribitol (Sigma-Aldrich, St. Louis, MO, USA) was added as an internal standard. Afterward, the aqueous sample was concentrated in a rotary vacuum centrifuge device (Labconco, Kansas City, MO, USA) for 4 h, and the resultant dried extracts were used for gas chromatography–mass spectrometry (GC-MS) analysis.
4
Metabolite Extraction and Derivatization Protocol
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The HOEC, DOK, SCC-9, SCC090 and Tca8113 cells were cultured to confluence in a petri dish and 2 × 106 cells were collected as previously described, with some modifications 11 . Briefly, cells were rinsed with distilled saline and then quenched thoroughly with 1 mL -20°C cold methanol (Sigma-Aldrich, St. Louis, MO, USA). The sediments were isolated by centrifugation with 6000 × g at 4°C for 10 min. The metabolites were extracted with 1 mL methanol by means of an ultrasonic cell disruptor (Scientz-950E, Ningbo, China) at a vibrational frequency of 360 W/40 kHz for 5 min. Then, a quantity of 10 µL ribitol (0.1 mg/mL) was added to each sample tube as an internal quantitative standard. The supernatant was harvested by centrifugation at 12,000 × g for 10 min and concentrated in a rotary vacuum centrifuge device (LABCONCO). The dried polar extracts were used for metabolite derivatization in GC-MS analysis. The dried residue was dissolved in 100 µL methoxyamine pyridine solution (20 mg/mL) and incubated at 37°C for 120 min in an incubator shaker. The mixture was treated with 100 µL N,O-Bis(trimethylsilyl)trifluoroacetamide (TMSTFA) with 1% trimethylchlorosilane (TMCS) and incubated at 37°C for 30 min. Every experiment was repeated by four biological replicates. In the case of biopsy samples, tissues of about 20 mg were homogenized in liquid nitrogen and supplemented with 1 mL methanol.
5
Metabolomic Analysis of Zebrafish Infection
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A total of 660 D. reiro were injected with saline (n = 60), Lev‐S (n = 300) or Lev‐R (n = 300), where the bacterial dose for Lev‐S and Lev‐R is 9 × 105 CFU per fish. The survival fish were collected and proceed for sample preparation for GC‐MS as previously described (Xu et al., 2019 ). D. rerio, 60 dpf, 0.22 ± 0.04g in weight and 2.52 ± 0.31 cm in length, were euthanized in ice slush (5 parts ice/1 part water, 0–4°C) for at least 10 min following cessation of gill movement and left in the ice water for a total of 20 min after cessation of all movement to ensure death by hypoxia following the guidelines of NIH (Reed and Jennings, 2011 ). D. rerio were rinsed with distilled water and then wiped thoroughly with sterilized gauze. Each fish was cut into five pieces on ice so the metabolites of the body fluids will diffuse out and then weighted. The appropriate volume of saline (100 μl 100 mg−1) was added according to the weight. After centrifugation at 3000 g at 4°C, 100 μl fluid was aliquoted for the following metabolomic analysis. Metabolites were extracted with 0.2 ml of cold methanol, containing 10 μl of 0.1 mg ml−1 ribitol (Sigma‐Aldrich) as an analytical internal standard. After centrifugation at 12 000 g for 10 min, the supernatant was concentrated in a rotary vacuum centrifuge device, LABCONCO. The dried polar extracts were used for GC‐MS analysis.
6
Metabolomic Analysis of Bacterial Samples
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Metabolomic samples were prepared
according to a previous method. Briefly, stationary-phase bacteria
were diluted 1:100 with fresh LB medium and recultivated until an
OD600 of 1.0. An aliquot of 10 mL of the cultures was then
collected and suspended in 1 mL of precooled methanol (−80
°C) for the extraction of cellular metabolites, with 10 μL
of 0.1 mg/mL ribitol (Sigma-Aldrich) serving as an internal standard.
Cells were then lysed by sonication for 5 min over an ice-water mixture
and centrifuged at 12,000 rpm at 4 °C for 10 min. Following this,
500 μL of supernatants were removed and dried using a rotary
vacuum centrifuge device (Labconco) at 37 °C. A total of 80 μL
of 20 mg/mL oxime hydrochloride (Sigma-Aldrich) in pyridine was added
to protect the carbonyl moieties of the samples through methoximation
for 90 min at 37 °C. After that, 80 μL of N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA,
Sigma-Aldrich) was used for derivatization at 37 °C for 30 min.
The obtained samples were detected by an Agilent 7890A GC and an Agilent
5975C inert XL mass selector (Agilent Technologies).
according to a previous method. Briefly, stationary-phase bacteria
were diluted 1:100 with fresh LB medium and recultivated until an
OD600 of 1.0. An aliquot of 10 mL of the cultures was then
collected and suspended in 1 mL of precooled methanol (−80
°C) for the extraction of cellular metabolites, with 10 μL
of 0.1 mg/mL ribitol (Sigma-Aldrich) serving as an internal standard.
Cells were then lysed by sonication for 5 min over an ice-water mixture
and centrifuged at 12,000 rpm at 4 °C for 10 min. Following this,
500 μL of supernatants were removed and dried using a rotary
vacuum centrifuge device (Labconco) at 37 °C. A total of 80 μL
of 20 mg/mL oxime hydrochloride (Sigma-Aldrich) in pyridine was added
to protect the carbonyl moieties of the samples through methoximation
for 90 min at 37 °C. After that, 80 μL of N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA,
Sigma-Aldrich) was used for derivatization at 37 °C for 30 min.
The obtained samples were detected by an Agilent 7890A GC and an Agilent
5975C inert XL mass selector (Agilent Technologies).
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