Samples of flesh, liver, brain, midgut, pyloric caeca from three fish per tank were prepared as pooled homogenates (n = 3 per treatment) whereas faecal samples were analysed on a tank basis (n = 3 per treatment). Total lipid was extracted from approximately 1 g of sample by homogenising in chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK), and content determined gravimetrically [28 (link)]. Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalysed transesterification at 50°C for 16 h [29 ], and FAME extracted and purified as described previously [30 (link)]. FAME were separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), on-column injector and a flame ionisation detector. Data were collected and processed using Chromcard for Windows (version 2.01; Thermoquest Italia S.p.A., Milan, Italy). Individual FAME were identified by comparison to known standards and published data [30 (link)].
Ultra turrax
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Germany
The Ultra-Turrax is a high-speed homogenizer designed for the rapid and efficient dispersion and emulsification of liquid and solid samples. It utilizes a high-speed rotor-stator system to generate shear forces that break down and mix materials into a homogeneous suspension.
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Lab products found in correlation
Zb wax column, by Phenomenex (4 mentions)
Fisons gc 8160, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
No 1 filter paper, by Cytiva (1 mentions)
Cp wax 52 cb, by Agilent Technologies (1 mentions)
Autosystem xl, by PerkinElmer (1 mentions)
M mlv rt, by Promega (1 mentions)
Ultra pure phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Triglyceride assay kit, by Abcam (1 mentions)
Cryogenic tubes, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rna nano chip, by Agilent Technologies (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
19 protocols using ultra turrax
1
Lipid Extraction and Fatty Acid Profiling of Fish Tissues
2
Extraction and Quantification of Lipids from Pork
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Total lipid of flesh
was extracted from the NQC (n = 6) according to Folch
et al.20 (link) Briefly, approximately 1 g of
homogenized NQC was extracted with 20 mL of chloroform/ methanol (2:1,
v/v) by blending with a tissue disrupter (Ultra-Turrax; Fisher Scientific,
Loughborough, UK) for 20 s before being left on ice for 1 h. The solution
was then partitioned by the addition of 5 mL of KCl solution (1%,
w/v), vortex mixed, then followed by centrifugation to clarify the
biphasic system. The upper aqueous layer was removed and discarded
with the lower chloroform layer filtered through a Whatman no. 1 filter
paper and solvent evaporated under a stream of oxygen-free nitrogen
before vacuum desiccation overnight. Total lipid was quantified gravimetrically
and resuspended to form a stock solution of 10 mg/mL in chloroform/methanol
(2:1, v/v) containing 0.01% (w/v) butylated hydroxytoluene as antioxidant.
A 1:80 dilution was used for neutral lipid, a 1:2 dilution for positive
mode phospholipid, and a 2.5 times concentration for negative mode
phospholipid analyses. Samples were injected in the chloroform/methanol
solution.
was extracted from the NQC (n = 6) according to Folch
et al.20 (link) Briefly, approximately 1 g of
homogenized NQC was extracted with 20 mL of chloroform/ methanol (2:1,
v/v) by blending with a tissue disrupter (Ultra-Turrax; Fisher Scientific,
Loughborough, UK) for 20 s before being left on ice for 1 h. The solution
was then partitioned by the addition of 5 mL of KCl solution (1%,
w/v), vortex mixed, then followed by centrifugation to clarify the
biphasic system. The upper aqueous layer was removed and discarded
with the lower chloroform layer filtered through a Whatman no. 1 filter
paper and solvent evaporated under a stream of oxygen-free nitrogen
before vacuum desiccation overnight. Total lipid was quantified gravimetrically
and resuspended to form a stock solution of 10 mg/mL in chloroform/methanol
(2:1, v/v) containing 0.01% (w/v) butylated hydroxytoluene as antioxidant.
A 1:80 dilution was used for neutral lipid, a 1:2 dilution for positive
mode phospholipid, and a 2.5 times concentration for negative mode
phospholipid analyses. Samples were injected in the chloroform/methanol
solution.
3
Fatty Acid Profiling in Fish Tissues
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Samples of flesh, liver, brain, head kidney, midgut and hindgut from three fish per tank were prepared as pooled homogenates (n = 3 per treatment) and total lipid extracted from 1 g by homogenizing in chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK), and content determined gravimetrically [17 (link)]. Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalyzed transesterification at 50°C for 16 h [18 ], and FAME extracted and quantified by a gas chromatograph (AutoSystem XL, Perkin Elmer, Waltham, MA) with Total Chrom Version 6.3.1 software. The system was equipped with an auto-injector (1 μl, inlet temperature 250°C) and a flame ionisation detector (FID, 280°C). The temperature program for the oven was 90°C for 1 min, then raised to 150°C at 30 min-1 and finally raised to 225°C at 3°C min-1 and held for 7 min. Helium was used as the carrier gas and a fused silica capillary column coated with a chemically bonded polyethylene glycol (CP-Wax 52CB, 25 m × 0.25 mm i.d; Varian, Palo Alto, CA) was used. Individual methyl esters were identified by comparison with known standards and a well-characterized fish oil, and also by reference to published data [19 ].
4
Fatty Acid Profiling in Fish Samples
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Samples of whole bodies and NQC from five fish per tank were prepared as pooled pen homogenates (n = 3 per diet). Total lipid was extracted from approximately 1 g of sample by homogenising in chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK), and content determined gravimetrically (Folch et al., 1957) . Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalysed transesterification at 50 °C for 16 h (Christie, 2003) , and FAME extracted and purified as described previously (Tocher and Harvie, 1988) .
FAME were separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), on-column injector and a flame ionisation detector. Data were collected and processed using Chromcard for Windows (version 2.01; Thermoquest Italia S.p. A., Milan, Italy). Individual FAME were identified by comparison to known standards and published data (Tocher and Harvie, 1988) .
FAME were separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), on-column injector and a flame ionisation detector. Data were collected and processed using Chromcard for Windows (version 2.01; Thermoquest Italia S.p. A., Milan, Italy). Individual FAME were identified by comparison to known standards and published data (Tocher and Harvie, 1988) .
5
Fatty Acid Profiling in Fish Tissues
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Samples of faeces, muscle (flesh), liver, gills, anterior intestine and brain from three fish per tank were prepared as pooled homogenates (n = 3 per treatment) and total lipid extracted by homogenising in chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK), with content determined gravimetrically [26 ]. Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalysed transesterification at 50 °C for 16 h [27 ], and FAME extracted and purified as described previously [28 (link)]. FAME were separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), on-column injector and a flame ionisation detector. Data were collected and processed using Chromcard for Windows (version 2.01; Thermoquest Italia S.p.A., Milan, Italy). Individual FAME were identified by comparison to known standards and published data [28 (link)] and results expressed as mole percentage.
6
Fatty Acid Profiling in Fish Samples
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Samples of whole bodies and NQC from five fish per tank were prepared as pooled pen homogenates (n = 3 per diet). Total lipid was extracted from approximately 1 g of sample by homogenising in chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK), and content determined gravimetrically (Folch et al., 1957) . Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalysed transesterification at 50 °C for 16 h (Christie, 2003) , and FAME extracted and purified as described previously (Tocher and Harvie, 1988) .
FAME were separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), on-column injector and a flame ionisation detector. Data were collected and processed using Chromcard for Windows (version 2.01; Thermoquest Italia S.p. A., Milan, Italy). Individual FAME were identified by comparison to known standards and published data (Tocher and Harvie, 1988) .
FAME were separated and quantified by gas-liquid chromatography using a Fisons GC-8160 (Thermo Scientific, Milan, Italy) equipped with a 30 m × 0.32 mm i.d. × 0.25 μm ZB-wax column (Phenomenex, Cheshire, UK), on-column injector and a flame ionisation detector. Data were collected and processed using Chromcard for Windows (version 2.01; Thermoquest Italia S.p. A., Milan, Italy). Individual FAME were identified by comparison to known standards and published data (Tocher and Harvie, 1988) .
7
Gravimetric Lipid Extraction and FAME Analysis
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Total lipid content was determined gravimetrically after extraction of ~1 g tissue or diet by homogenizing in 20 or 36 volumes of ice-cold chloroform/methanol (2:1 v/v) respectively, using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK) according to Folch, Lees and Stanley (1957) . Non-lipid impurities were isolated by washing with 0.88% (w/v) KCl and the upper aqueous layer removed by aspiration and the lower solvent layer containing the lipid extract dried under oxygen-free nitrogen.
Fatty acid methyl esters (FAMEs) from total lipid were prepared by acidcatalyzed transmethylation at 50 o C for 16h (Christie, 1993) . FAME were extracted and purified as described previously (Tocher & Harvie, 1988) (Tocher & Harvie, 1988) . Data were collected and processed using Chromcard for Windows (Version 1.19; Thermoquest Italia S.p.A., Milan, Italy) .
Fatty acid content per g of tissue was calculated using heptadecanoic acid (17:0) as internal standard.
Fatty acid methyl esters (FAMEs) from total lipid were prepared by acidcatalyzed transmethylation at 50 o C for 16h (Christie, 1993) . FAME were extracted and purified as described previously (Tocher & Harvie, 1988) (Tocher & Harvie, 1988) . Data were collected and processed using Chromcard for Windows (Version 1.19; Thermoquest Italia S.p.A., Milan, Italy) .
Fatty acid content per g of tissue was calculated using heptadecanoic acid (17:0) as internal standard.
8
Lipid Extraction from Flesh and Liver
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Total lipid of flesh and liver samples was extracted using a modified Folch method (37) . Samples of homogenised NQC of approximately 1 g were weighed in duplicate (0•5-1 g) into 50-ml quickfit tubes and homogenised in 20 volumes of ice-cold chloroform-methanol (2:1, v/v) using an Ultra Turrax tissue disruptor (Fisher Scientific). Potassium chloride (0•88 %) was added to the homogenised samples, mixed and centrifuged at 1500 rpm for 7 min to separate different layers. The upper aqueous phase was removed by aspiration, and the bottom layer containing the lipid extract was filtered (Whatman No. 1) and transferred to pre-weighed tubes. The lower solvent phase was evaporated on a N evaporator and desiccated overnight before re-weighing. Chloroform-methanol (2:1, v/v) containing 0•01 % (w/v) butylated hydroxytoluene (BHT) was used to resuspend total lipid at a concentration of 10 mg/ml. Total lipids were stored under N at -20°C until subsequent analysis. The accepted variance in measured lipid content between samples was ±10 %.
9
Lipid Extraction from Tissue Samples
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Lipid was extracted from flesh samples using a modified method of Folch et al. [19] .
Briefly, samples were homogenised in 16 ml of chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK). Non-lipid impurities were removed by washing with 4 ml of 0.88 % aqueous KCl (w/v) and the upper aqueous layer removed by aspiration, and the lower solvent layer containing the lipid extract dried under oxygen-free nitrogen. Total lipid content was determined gravimetrically after overnight desiccation in vacuo.
Briefly, samples were homogenised in 16 ml of chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disrupter (Fisher Scientific, Loughborough, UK). Non-lipid impurities were removed by washing with 4 ml of 0.88 % aqueous KCl (w/v) and the upper aqueous layer removed by aspiration, and the lower solvent layer containing the lipid extract dried under oxygen-free nitrogen. Total lipid content was determined gravimetrically after overnight desiccation in vacuo.
10
Cytokine and Chemokine Quantification in Brain Tissue
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Brain tissue extracts were obtained from sham-operated and UCCA O mice at 24 hours after the ischemia and stored at -20°C. Thereafter, the brain tissue was homogenized in an extraction solution (100 mg of tissue per 1 mL of extraction solution) containing 0.4 mol/L NaCl, 0.05% Tween™ 20, 0.5% BSA, 0.1 mmol/L phenylmethyl sulfonil fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L EDTA, and 20 KI aprotinin, using Ultra-Turrax (Fisher Scientific, Pittsburgh, PA, USA). The brain homogenate was centrifuged at 3000×g for 10 min at 4°C, and the supernatant was collected and stored at -20°C. Concentrations of the cytokines TNF-a and IL-1b and of the chemokine CXCL1 were determined using ELISA. The brain tissue supernatants were assayed in an ELISA setup using commercially available antibodies, according to the manufacturer' s procedures (R&D Systems, Minneapolis, MN, USA).
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