The conventional Hct test, also known as a packed-cell volume (PCV) test, was performed on healthy RBC samples by the micro-hematocrit centrifugation method. A hematocrit capillary tube (Drummond Scientific Company) was filled with whole blood and capped on one end with a clay sealant. Then, the tubes were centrifuged at 17,000× g for 5 min in a Sorvall Legend Micro 17 (Thermo Scientific), and the Hct was read from the capillary tube once aligned with a provided chart and the result was round to the nearest half percent. The Hct measured by this method is denoted by a PCV subscript (HctPCV).
Sorvall legend micro 17
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sorvall Legend Micro 17 is a compact, high-performance benchtop centrifuge designed for a variety of laboratory applications. It features a maximum speed of 17,000 rpm and a maximum relative centrifugal force of 24,600 x g. The centrifuge is equipped with a brushless induction motor and an aerodynamic rotor design for quiet and efficient operation.
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Lab products found in correlation
Speed vacuum, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Metformin hydrochloride, by Fujifilm (1 mentions)
Brain heart infusion broth, by HiMedia (1 mentions)
Glycerol, by Avantor (1 mentions)
Brain heart infusion agar, by HiMedia (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
11 protocols using sorvall legend micro 17
1
Hematocrit Measurement by Micro-Centrifugation
2
Serum Metabolite Extraction Protocol
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Metabolite extraction from serum was achieved as previously34 (link). Briefly, all the serum samples were thawed on ice and mixed properly. 10 µl of each serum sample was taken in microfuge tube (1.5 ml), (Genaxy, Cat No. GEN-MT-150-C. S) and then 30 µl of chilled Methanol, (Merck, Cat.No.1.06018.1000) to the sample, vortexed briefly and then kept at − 20 ℃ for 60 min.
The sample was then centrifuged (Sorvall Legend Micro17, Thermo Fisher Scientific, Cat.No. Ligend Micro 17) at 10,000 rpm for 10 min. After centrifugation 27ul supernatant was collected in separate microfuge tube without disturbing the pellet and dried using Speed Vacuum, (ThermoFisher Scientific, Cat.No. SPD1030-230) at low energy for 30–35 min. Samples pellets were then re-suspended using 30 ul Methanol: water (1:1, water: Methanol) mixture for injection.
The sample was then centrifuged (Sorvall Legend Micro17, Thermo Fisher Scientific, Cat.No. Ligend Micro 17) at 10,000 rpm for 10 min. After centrifugation 27ul supernatant was collected in separate microfuge tube without disturbing the pellet and dried using Speed Vacuum, (ThermoFisher Scientific, Cat.No. SPD1030-230) at low energy for 30–35 min. Samples pellets were then re-suspended using 30 ul Methanol: water (1:1, water: Methanol) mixture for injection.
3
Serum Metabolite Extraction for LC-MS Analysis
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Extraction of metabolites from serum was done as previously described39 (link). Briefly, serum samples were thawed on ice and then mixed prior to extraction. For metabolite extraction, 10 µl of serum was aliquoted into a 1.5 ml microcentrifuge tube (Genaxy, Cat No. GEN-MT-150-C. S). To this, 30 µl of chilled methanol (Merck, Cat. No. 1.06018.1000) was added and briefly vortexed. This mixture was then kept at −20 °C for 60 min.
After the incubation, the sample was centrifuged (Sorvall Legend Micro17, Thermo Fisher Scientific, Cat. No. Ligend Micro 17) at 10,000 rpm for 10 min. Supernatant (27 µl) was then carefully aspirated into a fresh microfuge tube without disturbing the pellet. Speed vacuum (ThermoFisher Scientific, Cat. No. SPD1030-230) was employed at low energy for 30 min to dry the supernatant. This dried sample was either stored at −80 °C for later use or reconstituted immediately with 30 µl methanol: water (1:1) for LCMS injection.
After the incubation, the sample was centrifuged (Sorvall Legend Micro17, Thermo Fisher Scientific, Cat. No. Ligend Micro 17) at 10,000 rpm for 10 min. Supernatant (27 µl) was then carefully aspirated into a fresh microfuge tube without disturbing the pellet. Speed vacuum (ThermoFisher Scientific, Cat. No. SPD1030-230) was employed at low energy for 30 min to dry the supernatant. This dried sample was either stored at −80 °C for later use or reconstituted immediately with 30 µl methanol: water (1:1) for LCMS injection.
4
Protein Quantification by UV Spectrophotometry
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The protein content of samples, donated by Cp, treated under different temperature was determined by using UV Spectrophotometry (Cao and Huang, 2005 ). In detail, the samples were added into the 1 mL centrifuge tube, which were skimmed at 10000 rpm at 4°C for 25 min using a centrifuge (Sorvall Legend Micro17, Thermo Fisher Scientific, USA). The supernatant was diluted 10 times, the absorbance of the supernatant under 260 nm and 280 nm was measured by UV spectrophotometer (TU-1900, Beijing Pgeneral Co., Ltd., China). Cp was calculated by the followi
Cp was calculated by the following formula:
Cp was calculated by the following formula:
5
Dissolution of Compounds 1 and 7
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Ten milligrams of 1 and 7 was added into the 15 mL centrifuge tubes, then 10 mL of water was added, and the tubes were shaken on a vortexes (Shanghai Qite Analytical Instrument Co., Shanghai, China; QT-2) at 1000 rpm for 30 min at room temperature. Subsequently, the tubes were ultrasonic dissolved for a further 3 h in an ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Kunshan, China; KQ-300DE). After completion of dissolution, the suspension was centrifuged at 15 000 rpm (Thermo Fisher, Waltham, MA, USA, Sorvall Legend Micro 17) for 10 min to precipitate undissolved particles. Then, the solid and liquid phases were separated, and the undissolved solid was dried and weighed.
6
Extraction and Evaluation of Melanian Snail Meat Powder
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Green powders of MPh were prepared by the following method: briefly, melanian snail meat samples were warmed at 50°C for 10 min, mixed with 5 volumes of distilled water, and then reacted with Protamax (E/S=3.6 AU/g) at 50°C for 10 min with shaking at 300 rpm. The enzyme activities were then inhibited by heating in a water bath at 95°C for 15 min. Finally, the samples were centrifuged at 1,500 × g (Sorvall Legend Micro 17; Thermo Fisher Scientific Inc., Waltham, MA, USA) for 10 min, and the supernatants were completely lyophilized. The MPh samples thus obtained were stored at −20°C and protected against light and humidity until use. Some MPh samples were deposited in the herbarium of the Medical Research Center for Globalization of Herbal Formulation, Daegu Haany University (encoded as MPh2015Ku01). Metformin hydrochloride (Wako, Osaka, Japan) was used as a reference.
MPh dissolved in distilled water was administered orally, at doses of 125, 250 and 500 mg/kg, once a day for 84 days after 7 days of HFD adaptation. Metformin dissolved in distilled water was also administered orally at 250 mg/kg. The intact vehicle and diabetic control mice received an oral administration of equal volumes of distilled water.
MPh dissolved in distilled water was administered orally, at doses of 125, 250 and 500 mg/kg, once a day for 84 days after 7 days of HFD adaptation. Metformin dissolved in distilled water was also administered orally at 250 mg/kg. The intact vehicle and diabetic control mice received an oral administration of equal volumes of distilled water.
7
Bacterial Culture Preparation for Surface Sampling
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Listeria monocytogenes Scott A, Listeria innocua ATCC 51742, Bacillus pumilus ATCC 700814, and Escherichia coli ATCC 25922 strains were maintained at −80 °C in Brain Heart Infusion broth (HiMedia, Mumbai, India) with 20% (vol/vol) glycerol (Amresco, Radnor, PA, USA). Cultures were activated by streaking onto Brain Heart Infusion agar (HiMedia, Mumbai, India) and incubated aerobically at 37 °C overnight. Individual colonies were picked, and triplicate portions of 10 mL Brain Heart Infusion broth were inoculated, then aerobically cultured at 37 °C and allowed to reach a pre-determined OD600.
A 1.0 mL portion of culture was centrifuged at 5000× g for 5 min at room temperature (Sorvall Legend Micro 17, Thermo Scientific, Waltham, MA, USA), washed once with Phosphate Buffered Saline pH 7.2 (PBS, BD Diagnostics, Franklin Lakes, NJ, USA), and resuspended in an equal volume.
For the preparation of bacterial cultures for surface sampling, washed cultures of L. monocytogenes and B. pumilus were diluted to prepare cultures of L. monocytogenes containing 101, 102, and 103 Colony Forming Units (CFU)/mL, and B. pumilus containing 103 CFU/mL.
A 1.0 mL portion of culture was centrifuged at 5000× g for 5 min at room temperature (Sorvall Legend Micro 17, Thermo Scientific, Waltham, MA, USA), washed once with Phosphate Buffered Saline pH 7.2 (PBS, BD Diagnostics, Franklin Lakes, NJ, USA), and resuspended in an equal volume.
For the preparation of bacterial cultures for surface sampling, washed cultures of L. monocytogenes and B. pumilus were diluted to prepare cultures of L. monocytogenes containing 101, 102, and 103 Colony Forming Units (CFU)/mL, and B. pumilus containing 103 CFU/mL.
8
Cultivation and Preparation of Stentor coeruleus
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Stentor coeruleus were cultured in Pasteurized Spring Water, or PSW (132458, Carolina Biological Supply Company, Burlington, NC, USA), in Pyrex dishes in the dark at room temperature. The average diameter of Stentor was 400 μm. Algae was cultured under constant light in Tris-acetate-phosphate (TAP) media. We fed Stentor cells 1 mL of concentrated algae per 100 mL of Stentor culture every other day. To prepare 1 mL of concentrated algae, we spun down 2 mL of Chlamydomonas culture that was healthy (with a very green appearance) using a centrifuge (Sorvall Legend Micro 17, Thermo Fisher Scientific, Waltham, MA, USA) at 2× g, removed the TAP, washed with 2 mL of PSW, repeated the centrifugation step, and washed with 1 mL of PSW. Stentor cultures were fed two days prior to the wounding experiments. To collect cells for the experiments, healthy adult cells (~400 μm in diameter and dark green in color) were retrieved from the culture by pipetting under a stereoscope and into a glass vial.
9
Intracellular and Viral DNA Extraction
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A 144 ml sample of the same water used for eDNA extraction was centrifuged (17,000 xg, 15 min, room temperature, in a Sorvall Legend Micro 17 centrifuge, Thermo Scientific) and the cell pellet used for the extraction of the intracellular DNA (iDNA, Fig. 1), using the protocol described in Mutlu et al., 2008. To compare the iDNA obtained by centrifugation and filtration through 0.2 µm, 200 mL were filtered and DNA extracted using the DNeasy blood and tissue kit (Qiagen). 16S rRNA gene from both iDNA were partially amplified by PCR using 341F and 805R primers (Herlemann et al., 2011) , products were sequenced by Illumina Nextera 2 x 250 bp and analyzed using Qiime (Caporaso et al., 2010) .
Viral DNA (vDNA) was extracted from the viral pellets obtained after the ultracentrifugation step (see below) using the protocol described in Santos et al. (2010) , which included embedding of the viral particles in agarose plugs. Prior to viral DNA purification from agarose plugs, the size range of vDNA was checked by pulsed field gel electrophoresis. The lack of cellular contamination in the viral pellet was checked by PCR with archaeal 16S rRNA gene primers as previously reported (Ramos-Barbero et al., 2019) . All the extracted DNAs were also quantified by Qubit 2.0 (Invitrogen).
Viral DNA (vDNA) was extracted from the viral pellets obtained after the ultracentrifugation step (see below) using the protocol described in Santos et al. (2010) , which included embedding of the viral particles in agarose plugs. Prior to viral DNA purification from agarose plugs, the size range of vDNA was checked by pulsed field gel electrophoresis. The lack of cellular contamination in the viral pellet was checked by PCR with archaeal 16S rRNA gene primers as previously reported (Ramos-Barbero et al., 2019) . All the extracted DNAs were also quantified by Qubit 2.0 (Invitrogen).
10
Emulsion Stability Determination in Salad Dressings
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Emulsion stability was determined by measuring the mass (g) of oil separated from the salad dressing after centrifugation. The newly formulated salad dressing with cocoa butter and control were incubated at 50 °C for 24 h. Approximately 1 g of each sample was placed in the centrifuge tubes and centrifuged for 10 min at 865×g (SorvallTM LegendTM Micro 17; ThermoFisher Scientific) at room temperature to separate the top oil layer (12 (link)).
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