For hIL-10 detection, the supernatant of PK(15) and PK(15)-hIL10 was collected and centrifuged at 300 g for 10 minutes. The samples were lyophilized using Freezone Plus (Labconco, 7960040) and the protein concentration was determined with the Bradford assay (Bio-rad, 500-0006). Proteins (5 μg/well) were added 2 X Laemli sample buffer (Bio-rad, 1610737) and reduced. Samples were separated by electrophoresis (Life technologies™, B1000) on 4 %–12% Bis-Tris polyacrylamide gels (Invitrogen, NW04120BOX), and the bands were transferred to nitrocellulose membrane (Bio-rad, 1620115). The membranes were blocked in Dulbecco’s Phosphate Buffered Saline (Welgene, LB001-02) with 0.1% tween 20 (Sigma, P9416)/5% skim milk (BD, 232100) for 1 hour at room temperature. Primary and secondary antibodies were diluted at 1:1000 and 1:5000, respectively for blotting. Quantitation and imaging of western blots were done using LAS 3000 imaging system (Fuji), following the manufacturer's instructions.
Freezone plus
Manufactured by Labconco
Sourced in United States
The FreeZone Plus is a high-performance freeze dryer designed for laboratory use. It features a stainless steel chamber and a condensing capacity of up to 8 liters per 24 hours. The product has a temperature range of -50°C to +50°C and can accommodate various sample sizes and configurations.
Automatically generated - may contain errors
Lab products found in correlation
Allegra 25r centrifuge, by Beckman Coulter (2 mentions)
Dulbecco s phosphate buffered saline, by Welgene (1 mentions)
Laemli sample buffer, by Bio-Rad (1 mentions)
B1000, by Thermo Fisher Scientific (1 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Mdf u53v, by Sanyo (1 mentions)
St 8r, by Thermo Fisher Scientific (1 mentions)
Pal autosampler, by Shimadzu (1 mentions)
Polyvinyl alcohol (pva), by Thermo Fisher Scientific (1 mentions)
Spectra por, by Repligen (1 mentions)
Cellulose acetate membrane filter, by Avantor (1 mentions)
Mini beadbeater, by Biospec (1 mentions)
Thermomixer c, by Eppendorf (1 mentions)
11 protocols using freezone plus
1
Quantitative Analysis of hIL-10 Expression
2
Antimicrobial Bacterial Cellulose Aerogels
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Biocomposites based on the BC aerogels and SF were obtained using the freeze-drying of hydrogels from native bacterial cellulose (NBC) and oxidized bacterial cellulose (OBC) with an OD of 1.44%. To impart the antimicrobial properties, the antibiotic SF, at concentrations of 500 μg/g, was added to the hydrogel. To obtain the aerogels, the hydrogels were frozen in a low-temperature cooler MDF-U53V (SANYO, Japan) at −50 °C for 24 h, and further dried on a FreeZone Plus freeze-dryer lyophilizer (Labconco, USA). The obtained aerogels were cut into round pieces with a diameter of 10 mm, weighing 0.002 g and containing 100 μg of SF. The final composites were identified as NBC/SF100, and OBC/SF100.
3
Defatting and Aqueous Extraction Protocol
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The
powdered samples were defatted using Soxhlet extraction with hexane
at 65 °C to ensure no exogenous lipids’ interference in
further experiments. The defatted samples were dried in an oven set
at 50 °C and further used to prepare the water extract in a 1:10
(w/v) ratio. The samples were kept in a shaker for 24 h at room temperature.
The extracts were filtered through muslin cloth followed by Whatman
no.1 filter paper, and finally, the filtrate was centrifuged at 5000
rpm for 10 min (Thermo scientific ST 8R). The obtained clear filtrates
were freeze-dried at −40 °C (FreeZone Plus, LABCONCO,
USA) and stored at −20 °C till further use.
For
the experiments, 50 mg extracts were dissolved in 1 mL of distilled
water to get the stock concentration of 50000 ppm. From this stock
solution, the working concentrations of 12.5, 25, 50, and 100 ppm
were prepared.
powdered samples were defatted using Soxhlet extraction with hexane
at 65 °C to ensure no exogenous lipids’ interference in
further experiments. The defatted samples were dried in an oven set
at 50 °C and further used to prepare the water extract in a 1:10
(w/v) ratio. The samples were kept in a shaker for 24 h at room temperature.
The extracts were filtered through muslin cloth followed by Whatman
no.1 filter paper, and finally, the filtrate was centrifuged at 5000
rpm for 10 min (Thermo scientific ST 8R). The obtained clear filtrates
were freeze-dried at −40 °C (FreeZone Plus, LABCONCO,
USA) and stored at −20 °C till further use.
For
the experiments, 50 mg extracts were dissolved in 1 mL of distilled
water to get the stock concentration of 50000 ppm. From this stock
solution, the working concentrations of 12.5, 25, 50, and 100 ppm
were prepared.
4
Peptide Conjugation and Purification
5
Algal Lipid Extraction Protocol
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The algal cells cultured for 5 days were collected by centrifugation at 4°C, 3,000 g for 5 min, and dried for 48 h in a vacuum freezing drier (Labconco FreeZone Plus, America). Total lipid content in dried cells was determined with a Chloroform-methanol method (Bligh and Dyer, 1959 (link)). A sample of 50 mg dried pellets was mixed with 3 mL chloroform - methanol (2:1, v/v) in a 15 mL tube, and then sonicated for 10 min in an ice-water bath. The supernatant was gathered into a 15 mL pre-weight glass tube by centrifuging at 6,800 g for 10 min. The processes of the above sonication and centrifugation steps were repeated twice for completely extracting lipids. All gathered supernatants were dried at 65°C in an oven. The lipid content was exhibited as DCW percentage (% DCW). The extracted lipids were used for further analyzing fatty acid composition.
6
Nanoprecipitation Synthesis of PLGA Nanoparticles
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NPs were prepared using nanoprecipitation.33 (link),43 (link) Briefly, 1 mg Nim (BioVision Incorporation) and 9 mg PLGA (50:50, inherent viscosity range 0.15–0.25 dL/g; Durect, Pelham, AL, USA) were dissolved in 1 mL acetone. Ten mL of a 1.5% w:v solution of polyvinyl alcohol (Alfa Aesar, Ward Hill, MA, USA) was taken in a beaker and the drug–polymer solution was added in a controlled manner (0.1 mL/min). The mixture was stirred for 5 hours at 500 rpm on a magnetic stirrer to evaporate acetone. The resulting suspension of NPs was centrifuged at 25,000 g (Allegra 25R centrifuge; Beckman Coulter, Brea, CA, USA) for 1 hour at 8°C. The NP pellet was suspended in deionized (DI) water after discarding the supernatant and centrifuged again in the same conditions. The resulting pellet was suspended in 3 mL DI water by vortexing. The suspension was transferred to a clean and dry amber-colored bottle, kept at −80°C for 2 hours, and then lyophilized overnight (FreeZone Plus; Labconco). Blank NPs were also prepared similarly by adding 10 mg PLGA to 1 mL acetone without the drug. NP preparation was performed in triplicate under light-protected conditions.
7
Alginate-Derived Aldehyde (ADA) Synthesis
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ADA was produced according to the method developed by Sarker et al. [17 (link)]. For this purpose, 5 g of sodium alginate (Art. No. 71238) was dissolved in 25 mL ethanol (99.8%), and 1.605 g sodium periodate was dissolved in 25 mL double distilled water. The sodium periodate solution was then added dropwise to the alginate–ethanol suspension under light exclusion with constant stirring (250 RPM). For the oxidation reaction, stirring was carried out for 6 h under the exclusion of light (i.e., the beaker was wrapped with aluminum foil). The reaction was stopped with 5 ml ethylene glycol, and then stirring was continued for 30 min. The ADA was dialyzed for 7 days against double distilled water to remove any remaining sodium periodate using the dialysis system Spectra/Por (Repligen, Boston, MA, USA) with standard RC dialysis membranes (6–8 kD MWCO). The water was changed twice a day. After dialysis, the ADA was dried in a lyophilization FreeZone Plus (Labconco, Kansas City, MO, USA) for another seven days. A 5% w/v ADA-solution was prepared by dissolving ADA in double-distilled water and stirred at 250 RPM overnight.
8
Fabrication of QCT-loaded Polymeric Micelles
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QCT-containing MPMs were fabricated by thin-film hydration method as described earlier, with some modifications.46 (link),47 Briefly, various polymers of 10 mM concentration at different proportions with QCT were dissolved in ethanol (Alfa Aesar). The solvent was removed under low pressure, in a rotary vacuum evaporator set at a temperature of 40°C for 1 h, to get a thin film of a mixture of QCT and polymers. To remove any residual ethanol, the thin film was subject to further drying under vacuum at room temperature for 24 h. Then, 3 mL of water was added to the thin film and shaken at room temperature to form micelles. The resultant micelles were then centrifuged at 10,000 rpm for 10 min (Allegra™ 25R Centrifuge; Beckman Coulter, Brea, CA, USA) followed by filtration of the supernatant using a 0.2 µm cellulose acetate membrane filter (VWR International, Radnor, PA, USA) to remove the unincorporated QCT aggregates. The mixed micellar solution was kept at −80°C for 2 h and then lyophilized (FreeZone® Plus™; Labconco Corporation, Kansas City, MO, USA) for 18 h. Similarly, empty mixed micelles (without QCT) were also fabricated.
9
Leaf Metabolite Extraction for NMR Analysis
10
Egg quality assessment under varied humidity and temperature
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A total of 290 eggs with an average weight of 53 g, and laid by Hy-Line white hens kept in the free-range system were collected in the 32 nd week of laying. An aqueous preparation containing H 2 O 2 (1.5%) and silver ions (0.015%) was used for the tests which would not cause an elastic deformation of eggshells (Tomczyk et al., 2018a) . The eggs were sanitised by immersion in 3% aqueous solution at 10°C for 5 min. Next, they were dried at 15°C, placed in sterile egg boxes, and separately stored according to different treatments; humidity (35, 65, 95% RH) and temperature (8, 20°C) for 4 w. Eggs were collected at the start of the experiment, and after 7, 14, and 21 d. In each sampling set, 58 eggs were collected. Next, egg white samples were freeze-dried (FreeZone Plus, Labconco, Kansas City, MO), and the shells were dried in a laboratory dryer at 50°C.
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