CRFK cells (2×106) were seeded in 10 cm dishes and incubated overnight. On the following day, the cells were infected with FCV at 0.2 MOI for 1 h and the supernatant was collected 5 h.p.i. and spun at 500 g at 4°C (Micro 22R, Hettich) for 30 min. After centrifugation, supernatant was filtered through a 0.2 μm Millex filter. The FCV particles were removed via precipitation by adding 0.2 M solid NaCl and 10% PEG3350 and incubated overnight at 4°C in constant rotation. Next, the samples were first centrifuged for 60 min at 500 g (Micro 22R, Hettich) at 4°C and then at 13,400 g for 4 h 30 at 4°C using an SW41Ti rotor (Beckman). The supernatant was then UV inactivated at a wavelength of 254 nm for 4 min (three times) using a crosslinker (Stratalinker® UV Crosslinker), filtered through a 0.2 μm Millex filter and stored in −20°C until use.
Micro 22r
Manufactured by Hettich
Sourced in Germany
The Micro 22R is a general-purpose refrigerated centrifuge designed for a wide range of laboratory applications. It features a compact footprint and can accommodate sample volumes up to 400 mL. The unit is equipped with a brushless induction drive motor and offers speed control up to 22,000 rpm, with a maximum RCF of 30,237 x g. The unit can maintain temperatures from -20°C to 40°C, ensuring precise temperature control for sensitive samples.
Automatically generated - may contain errors
5 protocols using micro 22r
1
Purification and Inactivation of FCV
2
Ultrasound-Assisted Extraction of Phytochemicals
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The flowers were ground in a coffee grinder for 5 min and then passed through a 200-µm Retsch sieve. WMF powder was measured (1 g) and mixed with the solvent (10 mL) in glass tubes with stoppers. Ultrasonication (US) was accomplished using an ultrasonic bath (Elma Transsonic 700/H, Singen, Germany) for 10, 30 and 50 min. The homogenates were centrifuged (Hettich, Micro 22R, Andreas Hettich GmbH and Co., Tuttlingen, Germany) for 10 min at 5000 rpm and the resulting supernatant was separated. Consecutively, the solvent of the crude extract was separated under vacuum at 45 °C using a rotary evaporator (Hei-VAP, Heidolph Instruments GmbH and Co., Schwabach, Germany) and the resulting residue was taken up in water and lyophilized (Advantage 2.0, SP Scientific, Warminster, PA, USA). If not stated otherwise, the lyophilized extracts dissolved in 70% EtOH (10 mg/mL) were used to perform the analyses.
3
Plasma Separation from Rat Blood
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Blood samples were collected from the retro-orbital venous plexus of the rats in heparinized tubes after (0.5, 1, 2, 4, 5, 6, 8, 10 and 12 h) following drug dosing. The plasma samples were separated after centrifugation (Centrifuge, Hettich Micro 22 R, Germany) of the blood samples for 10 min at 5000 rpm and kept at −20°C until analysis.
4
Quantification of Allium Phytochemicals
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A weighed amount of 1.0 g of fresh bulbs and leaves of onion and garlic, and roots of wood garlic was macerated in a mortar and pestle with 9 ml methanol added. After 30 min of extraction with magnetic stirring at room temperature, the extracts were obtained by filtering through gauze.
Additionally, the extracts were centrifuged for 20 min, at 15 000 rpm and at 4 °C (Microcentrifuge, Hettich; Micro 22R). The supernatants were evaporated to dryness, and stored at -20 °C until analysis. Prior to analysis by the HPLC system, the dry sample extract was weighed and methanol added to prepare a concentration of 1 mg ml -1 .
All three extracts were prepared in parallel the same way.
The content of methanolic extract per 1 g fresh sample of Allium species is listed in Table 1.
Additionally, the extracts were centrifuged for 20 min, at 15 000 rpm and at 4 °C (Microcentrifuge, Hettich; Micro 22R). The supernatants were evaporated to dryness, and stored at -20 °C until analysis. Prior to analysis by the HPLC system, the dry sample extract was weighed and methanol added to prepare a concentration of 1 mg ml -1 .
All three extracts were prepared in parallel the same way.
The content of methanolic extract per 1 g fresh sample of Allium species is listed in Table 1.
5
Enzymatic Characterization of Fungal Substrate
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Approximately 2 g of lyophilized colonized substrate was mixed with 20 mL sodium acetate buffer (0.05 M, pH = 5.0) and agitated (100 rpm) for 1 h at room temperature. The crude extracts were recovered by filtration (Whatman No2, England) followed by centrifugation (10,000× g, 15 min, 4 ± 0.1 °C) (Hettich Micro22R, Hettich, Germany). Clear supernatants were stored at −20 ± 1 °C for further analysis of endoglucanase and laccase activities according to the method described by Philippoussis et al. [29 (link)]. One unit of endoglucanase was defined as the amount of enzyme producing 1 μmol of reducing sugar (glucose equivalent) in one minute, under the conditions assayed. The standard curve was obtained with glucose for CMC. Laccase was determined using syringaldazine as substrate. One unit of laccase was defined as the amount of enzyme required to produce a change in absorbance of 0.001 per minute, under the conditions assayed. At least triplicates were used for the determination of each enzymatic activity which were expressed as U/g of dry substrate.
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