For β-carotene extraction, 0.5 mL of the cultivation broth was transferred into a 2 mL microtube (Sarstedt, Numbrecht, Germany). Each sample was centrifuged at 10 000x g for 10 min, and the supernatant was removed. Next, 0.5 mL of 0.5–0.75 mm acid-washed glass beads were added to each tube, followed by the addition of 0.5 mL of ethyl acetate supplemented with 0.01% 3,5-di-tert-4- butylhydroxytoluene (BHT). The BHT was added to prevent carotenoid oxidation. The cells were disrupted using a Precellys R 24 homogenizer (Bertin Corp., Montigny-le-Bretonneux, France) in four cycles of 5500 rpm for 20 s. The tubes were placed on ice for 1 min in between each lysis cycle to cool down and avoid product degradation. After disruption, the cells were centrifuged for 10 min at 10 000 x g. For the quantification of β-carotene by HPLC, 100 μL of the solvent fraction was transferred to HPLC vials. For DCW measurements, 1 mL of the cultivation broth was transferred into a pre-weighed 2 mL microtube (Sarstedt, Numbrecht, Germany). The tubes were centrifuged at 10 000 x g for 10 min. The supernatant was removed and the samples were washed with 1 mL of sterile water. The tubes with biomass pellets were dried at 60°C for 96 h and weighed on an analytical scale.
2 ml microtube
Manufactured by Sarstedt
Sourced in Germany
The 2 mL microtube is a small laboratory container designed to store and transport small liquid samples. It has a volume capacity of 2 milliliters.
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Lab products found in correlation
Synergy mx, by Agilent Technologies (1 mentions)
Uv 1800, by Shimadzu (1 mentions)
Hpx 87h ion exclusion column, by Aminex (1 mentions)
Ri 101 refractive index detector, by Thermo Fisher Scientific (1 mentions)
Uv spectrophotometer, by Shimadzu (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Dynamag 2, by Thermo Fisher Scientific (1 mentions)
Dvx 2500 multi tube vortexer, by Avantor (1 mentions)
Dnase 1, by Promega (1 mentions)
Primescript rt reagent kit perfect real time, by Takara Bio (1 mentions)
Macconkey agar, by Neogen (1 mentions)
10 protocols using 2 ml microtube
1
β-Carotene Extraction Protocol
2
Carotenoid Extraction and Quantification
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At the end of cultivation, 1 mL of the cultivation broth was transferred into a 2 mL microtube (Sarstedt) for carotenoids extraction. Each sample was centrifuged at 10,000 × g for 5 min and the supernatant removed. To each tube, 500 μL of 0.5–0.75 mm glass beads were added. 1 mL of ethyl acetate supplemented with 0.01% 3,5-di-tert-4-butylhydroxy toluene (BHT) was also added to each tube. BHT was supplemented to prevent carotenoid oxidation.
Cells were disrupted using a Precellys®24 homogenizer (Bertin Corp.) in four cycles of 5500 rpm for 20 s. After disruption, the cells were centrifuged for 5 min at 10,000 × g. For total carotenoid measurement, the solvent fraction was moved to a 96-well plate and read in BioTek Synergy™ Mx microplate reader with full spectrum scan (230 nm–700 nm) with 5 nm interval. Absorbance value at 450 nm was used to quantify total carotenoids. For the measurements of individual carotenoids, the samples were further processed before HPLC analysis as below.
Cells were disrupted using a Precellys®24 homogenizer (Bertin Corp.) in four cycles of 5500 rpm for 20 s. After disruption, the cells were centrifuged for 5 min at 10,000 × g. For total carotenoid measurement, the solvent fraction was moved to a 96-well plate and read in BioTek Synergy™ Mx microplate reader with full spectrum scan (230 nm–700 nm) with 5 nm interval. Absorbance value at 450 nm was used to quantify total carotenoids. For the measurements of individual carotenoids, the samples were further processed before HPLC analysis as below.
3
Quantifying Biomass and Glucose in Fermentation
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The OD600 values were detected with UV-1800 Shimadzu UV spectrophotometer. For the dry cell weight (DCW), 3 mL of the fermentation broth was filtered through preweighed cellulose nitrate membranes (0.45 µm pore size, 47 mm circle) using a filtration unit with a vacuum pump. The filters were dried at 60 °C for 96 h and weighed on an analytical scale. For glucose quantification, 1 mL of cultivation broth was transferred into a 2 mL microtube (Sarstedt, Numbrecht, Germany). The tubes were centrifuged at 10,000× g for 5 min. The supernatant was removed, filtered, and used for quantification on HPLC. The analysis on HPLC analyzed 20 µL of the sample for 30 min using an Aminex HPX-87H ion exclusion column with a 5 mM H2SO4 flow of 0.6 mL/min. The column temperature was set to 30 °C, the reflective index was set at 45 °C, and the glucose was detected using a RI-101 Refractive Index Detector (Dionex, Sunnyvale, CA, USA).
4
Antibody-Coated Magnetic Bead Preparation
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For each sample to undergo the ‘bead procedure’ 50 µL (100 µL for PFP samples diluted by a factor of 500) of Dynabeads protein G 30 mg/mL (Life Technologies, Carlsbad, CA, USA) were transferred to a 2 mL microtube (Sarstedt, Nümbrecht, Germany) after resuspension of the bead stock solution by vortexing for 30 seconds. A DynaMag-2 (Life Technologies) magnet was used to separate beads from the solution, and the supernatant was removed. Aliquots of 10 µL (20 µL for PFP samples diluted by a factor of 500) of polyclonal anti-human ApoB-48/-100 antibodies 1 mg/mL (Meridian, Life Science, Inc., Memphis, TN, USA) were diluted in 200 µL of PBS with 0.02% Tween 20 (Bio-Rad, Hercules, CA, USA) and then incubated with the magnetic beads under rotation for 10 minutes, allowing for the Fc-region of the antibodies to bind to protein G on the beads. The antibody coated beads were separated from the solution, which was removed. No antibodies were detectable in this removed solution (i.e. they were bound to the beads) as determined with measurement of absorption at 280 nm after this incubation. The antibody coated beads were washed three times with 200 µL of PBS with 0.02% Tween 20 by gentle pipetting. The microtube containing the antibody coated magnetic beads and the PBS used for the third wash was placed on the magnet, and the solution was removed from the fixed beads.
5
3D BEMER Metabolomic Analysis
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For metabolome analysis, A549 cells were cultured for 24 h in 3D lrECM followed by BEMER therapy (~13 μT, 8 min; sham-treated cells served as control). After 1 h, cells were harvested with 200 μl pre-cooled 80% MeOH containing 4 recovery standards to monitor extraction efficiency. The extraction solvent and cellular material were transferred into a 2 ml microtube (Sarstedt, Nümbrecht, Germany). Then, the wells were washed with 200 μl extraction solvent, which was collected in the same microtube. The samples were immediately stored in -80°C until analysis.
6
Yeast Growth and Biomass Quantification
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For precultures, 2.5 mL of Y10P20D20 in 24-well plates with an air-penetrable lid (EnzyScreen, NL) was inoculated with single yeast clones and grown for 16–24 h at 30°C and 300 rpm agitation. The optical density at 600 nm (OD600) was measured with a VWR NanoPhotometer 7122. Culture volume corresponding to 0.1 OD600 units was inoculated into 2.5 mL of Y10P20D80 or other media formulations in a 24-deep-well plate, which was incubated for 72 h at 30°C with 300 rpm agitation. All cultivations were performed in biological triplicates. Dry cell weight (DCW) was measured at the end of cultivation by transferring 0.5 or 1 mL of culture broth to a pre-weighed 2-mL microtube (Sarstedt). The samples were centrifuged and the supernatant was removed. The cell pellets were then dried at 60°C for 72 h.
7
Intracellular and Extracellular ABA Quantification
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For quantification of intracellular ABA concentration, 1 mL of culture broth was transferred to a 2-mL microtube (Sarstedt). The samples were centrifuged at 16 000 g for 5 min, and the supernatant was removed. The cell pellets were resuspended in water, centrifuged at 16 000 g for 5 min, and the supernatant was removed. This washing step was repeated. One milliliter of acetonitrile and 500 µL of 0.212–0.3 mm acid-washed glass beads were added to each tube. Thereafter, the cells were disrupted with a Precellys® 24 homogenizer (Bertin Corp.) using four cycles at 5500 rpm for 10 s each. The samples were then shaken for 10 min in a DVX-2500 Multi-Tube Vortexer (VWR, USA) at room temperature. Lastly, the samples were centrifuged at 16 000 g for 5 min, and the supernatant was collected for analysis.
For quantification of extracellular ABA concentration, 1 mL of culture broth was centrifuged at 16 000 g for 5 min and the supernatant was collected. Some samples were diluted with water before analysis.
For quantification of extracellular ABA concentration, 1 mL of culture broth was centrifuged at 16 000 g for 5 min and the supernatant was collected. Some samples were diluted with water before analysis.
8
Soil Nucleic Acid Extraction Protocol
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Regarding nucleic acid extraction, 0.5 g (wet weight) of soil was collected in a 2-mL microtube (Sarstedt, Nümbrecht, Germany) with 0.7 g of zirconium beads (diameter, 0.1 mm), frozen by liquid N2, and stored at −80°C. Total nucleic acids were extracted from soil, and DNA was digested with DNase I (Promega, Madison, WI, USA) according to the procedure described by Murase et al. (19 (link)). RNA dissolved in RNase-free TE buffer was stored at −80°C. The complete digestion of DNA in RNA samples was confirmed by PCR using the bacterial universal primer set 357f-GC/517r (21 (link)) in the absence of reverse transcriptase. Nucleic acid mixtures without the DNase treatment were used as DNA samples for the clone library analysis on day 0 and a DGGE analysis (see Supplemental document ). cDNA was synthesized from each RNA sample using the PrimeScript® RT reagent Kit (Perfect Real Time) (Takara, Otsu, Japan) with a random 6-mer according to the manufacturer’s instructions.
9
Preparation and Storage of Blood Samples
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Remaining whole blood samples were centrifuged at 2000 × g at 4°C for 20 minutes. Plasma and serum were removed into aliquots in 2 mL microtubes (Sarstedt Ltd.) and stored at −70°C. For vitamin C stabilization, 300 μL of plasma or serum was added to 300 μL of 10% w/v metaphosphoric acid.
After removal of plasma, washed erythrocytes were prepared by adding an approximate volume of normal saline to equal the original whole-blood volume in the tube, gentle inversion, and centrifugation at 2000 × g at 4°C for 10 minutes. The supernatant was removed and the process was repeated twice more (20 ). Washed erythrocytes were stored at −70°C until analysis.
Table 1 Supplemental Methods . Contemporaneous quality control and quality assurance data are available (13 ).
After removal of plasma, washed erythrocytes were prepared by adding an approximate volume of normal saline to equal the original whole-blood volume in the tube, gentle inversion, and centrifugation at 2000 × g at 4°C for 10 minutes. The supernatant was removed and the process was repeated twice more (20 ). Washed erythrocytes were stored at −70°C until analysis.
10
Isolation and Freezing of E. coli
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From each sample, fecal material was streaked onto MacConkey agar (Neogen, Michigan, USA) using a sterile cotton-tipped swab and incubated overnight at 37 °C. Five lactose-fermenting (bright pink) colonies with typical E. coli morphology were randomly selected from the MacConkey agar plate. These colonies were subcultured on horse blood agar plates (Neogen, Michigan, USA), incubated overnight at 37 °C, and tested for production of tryptophanase (indole) using the spot indole test (Miller and Wright 1982 (link)). Lactose- and indole-positive isolates with typical colony morphology (bright pink on MacConkey agar, blue in spot indole test and single-colony types) were considered E. coli. Confirmed isolates of E. coli were transferred to 2-mL microtubes (SARSTEDT, Nümbrecht, Germany) containing 0.5 mL serum broth supplemented with 15% glycerol. The microtubes were placed in a freezer at − 80 °C. One of the five frozen isolates of E. coli from each calf sample was selected at random and sent frozen (on dry ice) to the National Veterinary Institute, Uppsala, Sweden. Directly upon arrival, isolates were transferred to a freezer at − 20 °C and stored until further testing.
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