Mealworm larvae were sampled each week for analysis. Mealworms were fasted for 24 h and then euthanized by freezing at −40 °C for 10 min. For proximate analyses, mealworms were finely ground using a blender. For antioxidant activity assays, one gram of mealworm larvae was homogenized using a Polytron PT 3000 homogenizer (Kinematica AG, Luzern, Switzerland) in 10 mL of 70% (v/v) ethanol aqueous solution until no large pieces were visible (approximately 20 s). The homogenized samples were centrifuged using a Biofuge Stratos centrifuge (Kendro Laboratory Products, Asheville, NC, USA) at 5000× g for 10 min at 4 °C. The supernatants were collected for the analyses.
Polytron pt 3000
Manufactured by Kinematica
Sourced in Switzerland
The Polytron PT 3000 is a laboratory homogenizer for the mechanical disruption of biological samples. It is designed to effectively disperse, emulsify, and homogenize a variety of materials, including tissues, cells, and suspensions. The Polytron PT 3000 utilizes a high-speed rotor-stator system to generate shear forces for efficient sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
V 530, by Jasco (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Butylated hydroxytoluene bht, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Gf c filter, by Cytiva (1 mentions)
L 7420 uv detector, by Hitachi (1 mentions)
L 7100 pump, by Hitachi (1 mentions)
Prostaglandin e2 express eia kit, by Cayman Chemical (1 mentions)
8 protocols using polytron pt 3000
1
Antioxidant Potential of Mealworms
2
Malondialdehyde Quantification in Meat
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Thiobarbituric acid-reactive substances were measured for determination of malondialdehyde (MDA) levels according to the method described by Ke, Ackman, Linke, & Nash (1977) and modified by Dal Bosco et al. (2009) . A 5 g sample was taken from raw burgers and homogenized for 45 sec at 9000 rpm (Polytron PT 3000, Kinematica AG, Eschbach, Deutschland) with 10 mL of 7.5% trichloroacetic acid (TCA) and 0.1% diethylenetriaminepentaacetic acid (DTPA) in distilled water (final concentration).
The homogenized sample was centrifuged (10000 rpm for 10 min) (4235A CWS, ALC International, Milan, Italy) and filtered through Whatman number 1 filter paper, and 5 mL of the filtrate was mixed with 2.5 mL of 2-thiobarbituric acid (TBA) solution (0.288% in distilled water) in capped test tubes.
The tubes were vortexed and placed in a water bath at 95 °C for 45 min, then cooled under tap water. The absorbance was determined at 532 nm (V-530 Jasco International, Milan, Italy) against a blank containing TCA/DTPA solution instead of a sample extract. A calibration curve was plotted with TEP (1,1,3,3-tetraethoxypropane; 0-15 µM, final concentrations) to obtain the MDA concentration, and the results were expressed as mg of MDA per kilogram of fresh meat. All determinations were performed in triplicate.
The homogenized sample was centrifuged (10000 rpm for 10 min) (4235A CWS, ALC International, Milan, Italy) and filtered through Whatman number 1 filter paper, and 5 mL of the filtrate was mixed with 2.5 mL of 2-thiobarbituric acid (TBA) solution (0.288% in distilled water) in capped test tubes.
The tubes were vortexed and placed in a water bath at 95 °C for 45 min, then cooled under tap water. The absorbance was determined at 532 nm (V-530 Jasco International, Milan, Italy) against a blank containing TCA/DTPA solution instead of a sample extract. A calibration curve was plotted with TEP (1,1,3,3-tetraethoxypropane; 0-15 µM, final concentrations) to obtain the MDA concentration, and the results were expressed as mg of MDA per kilogram of fresh meat. All determinations were performed in triplicate.
3
Lipid Extraction and Analysis Protocol
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Minced samples (2.5 g) were homogenized using a Polytron PT 3000 (Kinematica®, Lucerne, Switzerland) for 30 s and added to flasks containing modified Folch’s solution (2:1 v/v chloroform:methanol; Fisher Scientific Co., Fair Lawn, NJ, USA) and 0.02% butylated hydroxytoluene (BHT; Sigma Chemical Co., St. Louis, MO, USA), to a final concentration of ~35 μg/mg fat [42 (link)]. Thereafter, the solution was passed through fluted Whatman #1 filter paper into 100-mL glass-stoppered graduated cylinders. Nonlipid substances were extracted by adding 10 mL of 0.88% (w/v) sodium chloride (Fisher Scientific) to the filtrate. A second extraction was conducted and the final volume (Vf) of the bottom lipid layer recorded. An aliquot of the bottom layer was transferred to dry, aluminum dishes of known weight and flushed with nitrogen gas. The dishes were weighed and placed in a desiccator overnight. Ratios of crude lipid (% w/w) were determined at the beginning and at the end of storage. Four replicate samples were measured in triplicate. The crude lipid content (%) was calculated as:
4
Isolating Endocrine and Exocrine Tissues
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Isolated endocrine (75–95% islet purity) and exocrine tissues were homogenized in ice-cold 0.32 M sucrose by hand using a Dounce glass homogenizer to a final concentration of 6 mg/ml. 50–100 mg INS-1 xenografts were homogenized using a Polytron tissue homogenizer (Polytron® PT 3000, Kinematica AG, Littau-Luzern, Switzerland) in ice-cold 0.32 M sucrose at a concentration of 6 mg/ml and then by hand using a Dounce glass homogenizer. Protein concentration was determined using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as standard. Aliquots of the homogenates were stored at −80 °C until used.
5
Radioligand Binding Assay for Islet Tissue
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Islets of Langerhans and exocrine tissue isolated from a non-diabetic deceased donor was obtained within the Nordic Network for Clinical Islet Transplantation Laboratory in Uppsala, Sweden. The islets (93% pure) and exocrine fractions were separately homogenized in ice-cold 0.32 M sucrose by hand using a Dounce glass homogenizer using a polytron tissue homogenizer (Polytron® PT 3000, Kinematica AG, Littau, Switzerland) in ice-cold 0.32 M sucrose at a concentration of 6 mg/ml and then by hand using a Dounce glass homogenizer. Aliquots of the homogenates were stored at − 80 °C until used.
Two milligrams of homogenized islets of Langerhans and exocrine tissue were separately incubated at RT for 30 min with 1 MBq [11C]AZ12204657 (corresponding to 5 nM) in 50 mM TRIS (pH 7.4) in a final incubation volume of 1 mL. Twenty micromolars of AZD3825 was added in a separate assay for determination of non-specific binding. The samples were filtered using a Brandel M-48 cell harvester with Whatman GF/C filter (presoaked with 50 mM TRIS) and washed four times with 3 mL 50 mM TRIS (RT). All samples were performed in triplicates. Filters were measured in a well counter (Uppsala Imanet AB, Uppsala, Sweden).
Two milligrams of homogenized islets of Langerhans and exocrine tissue were separately incubated at RT for 30 min with 1 MBq [11C]AZ12204657 (corresponding to 5 nM) in 50 mM TRIS (pH 7.4) in a final incubation volume of 1 mL. Twenty micromolars of AZD3825 was added in a separate assay for determination of non-specific binding. The samples were filtered using a Brandel M-48 cell harvester with Whatman GF/C filter (presoaked with 50 mM TRIS) and washed four times with 3 mL 50 mM TRIS (RT). All samples were performed in triplicates. Filters were measured in a well counter (Uppsala Imanet AB, Uppsala, Sweden).
6
BPSP HPLC Analysis Protocol
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For HPLC analysis, 0.1 g BPSP was weighed, deposited into a 10 mL-screw tube and homogenized with 5 mL 60% ethanol (Kinematica AG Polytron PT3000, Littau, Switzerland) at 15,000 rpm for 1 min. After heating the crew-capped tube in a water bath at 70 °C for 30 min with occassioal shaking, the tubes were centrifuged (Sigma Labrozentrifugen 2K15, Osterode am Harz, Germany) at 15,000 g for 15 min at 20 °C. The supernatant was membrane-filtered (0.45 μm) and stored at 4 °C for HPLC analysis.
For HPLC analysis, the procedure of Chang et al. (2006 (link)) was followed with minor modification. A dual pump (L-7100 pump, Hitachi Co., Tokyo, Japan) equipped with an UV-detector (L-7420 UV detector, Hitachi Co., Tokyo, Japan) run with an ODS column (Hypersil ODS column, 250 × 4.60 mm, 5 μm, Thermal Hypersil Ltd., Cheshire, UK). Two solvents, i.e., A: methanol and B: pure water were run with a gradient program initiated by 0 min: 0% A; 10 min: 30% A; 20 min: 100% A; 23 min: 100% A; 28 min: 0% A and 30 min: 0% A. The injection volume and monitoring wavelengh were 20 µL and 254 nm, respectively.
For HPLC analysis, the procedure of Chang et al. (2006 (link)) was followed with minor modification. A dual pump (L-7100 pump, Hitachi Co., Tokyo, Japan) equipped with an UV-detector (L-7420 UV detector, Hitachi Co., Tokyo, Japan) run with an ODS column (Hypersil ODS column, 250 × 4.60 mm, 5 μm, Thermal Hypersil Ltd., Cheshire, UK). Two solvents, i.e., A: methanol and B: pure water were run with a gradient program initiated by 0 min: 0% A; 10 min: 30% A; 20 min: 100% A; 23 min: 100% A; 28 min: 0% A and 30 min: 0% A. The injection volume and monitoring wavelengh were 20 µL and 254 nm, respectively.
7
Quantifying Kidney Prostaglandin E2 After I/R
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Kidney was harvested 48 hours after I/R treatment and homogenized in 100 mM phosphate buffer containing 1 mM EDTA and 10 μM COX inhibitor™ (pH 7.4) using a Polytron PT3000 (Kinematica AG). After centrifugation at 8,000 g for 10 minutes at 4°C, the obtained supernatant was used to assay prostaglandin E2 (PGE2) concentration using a Prostaglandin E2 Express EIA Kit (Cayman Chemical Co, San Diego, CA, USA).
8
Cellular Fractionation and Protein Analysis
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