The morphology of the soluble enzyme and immobilized CLEA was examined by scanning electron microscopy (SEM; Sigma 500 VP Field Emission-Scanning Electron Microscope, Zeiss, Oberkochen, Germany). Crude enzyme extract and CLEA produced under optimum conditions were prepared for SEM by freeze-drying for three consecutive days in a freeze-dryer (Modulyo 4K; Edwards, Burgess Hill, UK).
Modulyo 4k
Manufactured by Edwards Lifesciences
Sourced in United Kingdom
The Modulyo 4K is a piece of laboratory equipment designed for lyophilization, commonly known as freeze-drying. It has a temperature range of -80°C to +60°C and a chamber capacity of up to 4 square meters. The Modulyo 4K is used for the freeze-drying of various materials, including pharmaceuticals, biological samples, and other temperature-sensitive products.
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9 protocols using modulyo 4k
1
Enzyme Morphology Analysis via SEM
2
Biomimetic Matrix Coating for Cell Culture
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The tissue culture polystyrene (TCPS) surface was coated with specifically composed biomimetic matrix according to the protocol of Prasad Chennazy and Krishnan.13 (link) One milliliter of the composed matrix comprised 5 mg in-house isolated human cryoprecipitate (clottable fibrinogen and fibronectin), 0.2% gelatin (Sigma), and 100 μg hyaluronic acid (in-house purified and characterized14 (link)), 20 μg of released platelet growth factor (PGF) prepared per Resmi et al.,15 (link) 25 μg laminin V (Sigma), and 250 ng recombinant epidermal growth factor (EGF; Sigma). Briefly, matrix composite was clotted on thrombin adsorbed TCPS to get a thin fibrin layer, and dishes were lyophilized under sterile atmosphere using a freeze drier (Edwards, Modulyo 4K). The TCPS immobilized with composite fibrin matrix were then stored at 4°C to 6°C until use.
3
Biomimetic Fibrin-Polymer Composite Scaffold
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The terpolymer PLGC was synthesized using a reported procedure19 with a starting monomer (Sigma Chemicals) ratio of 70:10:20. For electrospinning, a well-characterized (data not shown) polymer solution (15% w/w) in dichloromethane was fed using a syringe pump (Holmarc Opto-Mechatronics Pvt. Ltd) at a flow rate of 3 mL/hour, and a voltage of 11 kV was applied (Zeonics Systech Defence and Aerospace Engineers Ltd.) to collect the fibers. The electrospun fiber matrices were dried under vacuum at room temperature for 24 hours, cut into patches of the required size for each experiment, and sterilized using ethylene oxide.
The biomimetic fibrin composite matrix was deposited on the polymer as described earlier.11 (link) The biomimetic matrix comprised cryoprecipitated human fibrinogen concentrate (10 mg/mL) and HA (50 μg/mL) prepared in-house as described previously.15 (link) Briefly, 100 μL/cm2 fibrin composite was layered on a sterile, thrombin-adsorbed scaffold, incubated for 30 min at 37°C, lyophilized (Edwards, Modulyo 4K), and stored at 4°C–6°C until use.
The biomimetic fibrin composite matrix was deposited on the polymer as described earlier.11 (link) The biomimetic matrix comprised cryoprecipitated human fibrinogen concentrate (10 mg/mL) and HA (50 μg/mL) prepared in-house as described previously.15 (link) Briefly, 100 μL/cm2 fibrin composite was layered on a sterile, thrombin-adsorbed scaffold, incubated for 30 min at 37°C, lyophilized (Edwards, Modulyo 4K), and stored at 4°C–6°C until use.
4
Reindeer Capture-Mark-Recapture Serum Collection
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Reindeer, 3‐ to 15‐year‐old females, were caught annually in late winter (late April to early May) using a net between two snowmobiles and restrained without immobilization drugs as part of a long‐term capture‐mark‐recapture study (Milner et al., 2003 (link); Omsjoe et al., 2009 (link); Stien et al., 2002 (link)). During capture, ear tags and neck marker straps on each reindeer were used to record the individual's identity, then live body mass was measured and pregnancy was diagnosed (Ropstad et al., 1999 (link)). Blood was collected from the jugular vein into plain vacutainers and centrifuged (2000 RCF, 10 min) within 12 h to retrieve serum. Serum samples were obtained from 1995 to 2012, excluding the years 2003 and 2010, resulting in a total of 16 sampling campaigns. Altogether 232 samples were collected from 182 individual reindeer, thus, some individuals were sampled only once and others up to three times during our study period. The number of samples per campaign ranged from 10–21, with the average value being 15. Serum samples were stored frozen at −80°C until freeze‐drying (48 h, −45°C, 8 mbar, Modulyo 4 K, Edwards).
5
Optimizing Hop Leaf Drying Techniques
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Humulus lupulus leaves from five hop genotypes (Table 1 ) grown under organic farming conditions were collected at harvest at the farm I Vizi del Luppolo (Cori, Italy; 41°63′46″ N-12°87′18″ E) and cold-transported to the Food Chemistry and Biotechnology laboratory at CREA Research Centre for Olive, Fruit and Citrus Crops (Rome, Italy). An aliquot of leaves (300 g) of each genotype was subjected to oven drying (OD) at 45 °C (air velocity: 0.6 ms−1, relative humidity < 0.5%, system power: 1.4 kW/h; model 600, Memmert GmbH + Co.KG, Schwabach, Germany). This type of drying and the temperature were chosen considering the possibility of exploiting the drying systems for hop cones that are generally present in these farms. The remaining part (300 g) was freeze-dried at −54 °C and 0.075 mbar (model Modulyo 4 K, Edwards, UK). Sample dehydration using all of the methods mentioned above was continued until about 8–10% final moisture content was reached. At the end of each drying treatment, samples were finely milled (sieve 0.5 mm), stored under vacuum and kept protected from light and moisture until analysis. Four replicates for each treatment were carried out.
6
Lyophilization of Zinc Nanoparticles
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The ZNPs of concentration 1 mg/mL and final volume 2 mL were poured into semi-stoppered glass vials with slotted rubber closures. ZNPs were initially frozen at −80 °C for 24 h followed by lyophilization using a Modulyo 4 K freeze-dryer (Edwards, Crawley, UK) at 0.09 mbar for 72 h. The temperature of the condenser surface was maintained at −60 °C ± 5 °C. After lyophilization, ZNPs were stored in a 4 °C chamber until future use.
7
Extraction and Characterization of A. satureioides Inflorescence Compounds
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Three extracts from A. satureioides inflorescences were prepared, including an aqueous extract, freeze-dried extract, and spray-dried extract. The plant : solvent ratio was 7.5 : 100 (w/v) for the three extracts. The aqueous extractive solution was prepared via decoction and then freeze-dried. The freeze-dried and spray-dried extracts were obtained by maceration of inflorescences in 80% ethanol (v/v). The extraction time was eight days, with occasional stirring [18 (link)]. The resulting extractive solution was filtered, and the supernatant was freeze-dried (frozen at −80°C and subsequently dried in a freeze-dryer (Edwards Modulyo 4K, Irvine, USA) at −60°C and pressure of −10−2 bar) or spray-dried (Spray Dryer Buchi B-290, with a two-component nozzle and current flow, under the following operating conditions: inlet temperature, 160 + 2°C; output temperature 140 + 2°C; feed rate, 3 mL/min; and spraying pressure, 2 bar [19 ]). The spray-dried extract contained 50% extractives, 33.4% colloidal silicon dioxide, and 16.6% polysorbate 80.
8
Fibrin Matrix for Cell Differentiation
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Fibrin matrix-based niche used for differentiation and signaling studies was prepared using a modified, well-established protocol38 (link). Pharmacopoeia grade fibrin sealant (Drug controller approved) was used for coating tissue culture polystyrene (TCPS, NUNC, Roskilder, Denmark).
Fibrinogen and thrombin were reconstituted using sterile distilled water. Thrombin (5 IU) was poured on to TCPS in a minimal volume to cover the TCPS surfaces. Incubation was done at 37 °C for 30 minutes and the thrombin was completely poured off after the incubation period. The thrombin adsorbed surface was layered with a thin layer of diluted fibrinogen (2 mg/ml). The clot formed was stabilized by incubating the coated TCPS at 37 °C for 30 minutes. The plates were frozen overnight at −80 °C. The plates were lyophilized (Modulyo 4 K, Edwards, UK) and stored at 4 °C in a sterile environment.
Fibrinogen and thrombin were reconstituted using sterile distilled water. Thrombin (5 IU) was poured on to TCPS in a minimal volume to cover the TCPS surfaces. Incubation was done at 37 °C for 30 minutes and the thrombin was completely poured off after the incubation period. The thrombin adsorbed surface was layered with a thin layer of diluted fibrinogen (2 mg/ml). The clot formed was stabilized by incubating the coated TCPS at 37 °C for 30 minutes. The plates were frozen overnight at −80 °C. The plates were lyophilized (Modulyo 4 K, Edwards, UK) and stored at 4 °C in a sterile environment.
9
Sour Cherry Pomace Drying Methods
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Single-cultivar pomaces (2.5 kg each one), from the juice processing of two different sour cherry varieties (a local "Morello or visciola" sour cherry selection recovered by Bianchi in Offagna AN, Italy, and Montmorency, namely BO and MM, respectively) grown under organic farming, were kindly provided by Italia Selvatica SRL Agricola Offagna, Italy. Pits, stems and other foreign materials were manually removed from pomaces, which were then stored at -20°C in low-density polyethylene bags until use. An aliquot of pomace (100g) was directly analysed and considered as control samples (CTRs) whereas (400g) of each variety was subjected to oven drying (OD) at 60° C for 24 h (air velocity: 0.6 ms -1 , relative humidity < 0.5%, system power: 1.4 kW/h); model 600, Memmert GmbH + Co.KG, Schwabach, Germany), and the remaining part (400g) was freeze-dried (FD) at -54°C and 0.075 mbar for 72h (model Modulyo 4K, Edwards, United Kingdom). Sample dehydration using all the methods mentioned above was continued up to 9% final moisture content was reached. At the end of each drying treatment, pomaces were finely milled (sieve 0.5 mm) and kept protected from light and humidity until analysis. Three replicates for each treatment were carried on.
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