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89 protocols using freezone 2

1

Solvent Extraction of Dry Plant Material

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Air-dry, powdered raw material (10 g) was extracted with 100 mL of solvent (ethanol:water; 60:40, v/v) in Büchi Extraction System B-811 (Büchi Labortechnik AG, Flawil, Switzerland). Soxhlet hot extraction with twenty-five cycles was used. In order to obtain sufficient amount of extracts, the extraction was repeated 10 times. Obtained extracts were filtered, concentrated using a rotary evaporator Büchi R200 (Büchi Labortechnik AG), frozen in −80 °C for 5 days, and finally lyophilized (Labconco Freezone 2.5, Labconco, Kansas City, MO, USA). Dry extracts were powdered and stored in dark vials, at 4 °C.
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2

Extraction and Drying of Larval Biomass

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The LMSN were kept frozen at -40 o C. Prior to performing the extractions, the larvae were washed in a steel sieve under running water, followed by two washes with distilled water. The excess water was removed with the aid of absorbent paper before weighing the wet biomass, which was approximately 218 g. Subsequently, the larvae were dried at 70 o C for 48 hours in an oven (Sanyo-Drying oven) (7) . The determination of dry weight was made after the temperature stabilized (30 minutes in the desiccator). For all the methods subsequently applied, 10 g of dried larvae sample were used. The humidity contained in the larvae was (on average) 66%. Five milliliters of all the aqueous fractions (supernatants) obtained from the extraction methods were collected for lyophilisation (Labconco FreeZone 25 ® , Labconco Corporation Kansas City, MO, USA). The dry weight of samples obtained was determined before storage for future analysis.
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3

Lyophilization and Ethanolic Extraction

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Five g of pulp and seeds, separately, were chopped and lyophilized (FreeZone 2.5 L Labconco, USA). The material was extracted (20% w/v) with a mixture of ethanol-distilled water (1:1 v/v) as solvent (at 25 °C, for two h in an ultrasonic bath). The extract was filtered, and the solvent was evaporated under vacuum at 45 °C. The extract was frozen and lyophilized, until a yield of 327.3 and 125.8 mg of dark brown gums (6.54 and 2.51%) from pulp and seeds, respectively.
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4

Freeze-dried Larval Biomass Extraction

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As LMSN foram mantidas congeladas a -40 o C. Para realizar as extrações, as larvas foram lavadas em tamis de aço em água corrente, seguidas de duas lavagens com água destilada. O excesso de água foi removido com o auxílio de papel absorvente, antes da pesagem da biomassa húmida, que foi cerca de 218g. Posteriormente, as larvas foram secas a 70 o C por 48 horas em estufa (Estufa-SanyoDrying) (7) . A determinação do peso seco foi realizada após a temperatura estabilizada (30 minutos no exsicador). Para todos os métodos aplicados a seguir, foram utilizados 10 g de amostra de larvas secas. A humidade contida nas larvas foi em média 66%. Foram coletados 5 mL de todas as frações aquosas (sobrenadante) obtidas pelos métodos de extração para liofilização (Labconco FreeZone 25®, Labconco Corporation Kansas City, MO, EUA), determinado o peso seco e armazenados para análise futura. As extrações foram realizadas em triplicado.
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5

Trehalose-Stabilized Nanoparticle Freeze-Drying

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The NPs in suspension form containing 1% trehalose as cryoprotectant was pre-cooled at −20 °C overnight. Upon pre-cooling, the samples were placed in the freeze dryer (Freezone 2.5, LABCONCO, Kansas city, MO, USA) at 6 Pa for 72 h maintained at −50 °C. These freeze-dried NPs were used for thermal studies and FTIR analysis.
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6

Physalis peruviana L. Bioactive Extracts

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The extracts analyzed in this study were obtained from the mature berries and leaves of Physalis peruviana L. purchased of different local retail shops from the city of Guaranda (Ecuador). The berries and healthy leaves were washed and triturated, in parallel also were lyophilized in a Freeze drier (Labconco Free Zone 2.5, USA), for their later processed. Fifteen grams of uvilla (berries and leaves) were put to maceration in 100 ml of 95% ethanol and 100 ml of distiller water at room temperatura for 6 d. According to the method established by Çakiret al. [6] for ethyl extract and Areiza et al. [16] for aqueous extract with so memodifications. In table 1, the treatments performed in this research are detailed where a DBCA was applied with factorial arrangement AxB: A (the type of Extract) and B (Parts of the vegetable).
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7

Olive Cultivar Diversity and Pest Infestation

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Samples, were collected in the middle of November 2015 from four commercial olive orchards with some of the most important cultivars grown in Southern Italy: Leccino, Ottobratica, Carolea, and Ciciarello. Orchards were located in Monopoli (Puglia, Southern Italy), and in Mileto, Filadelfia and Maierato (Calabria, Southern Italy), respectively (Table 1).
From each location, 5 biological replicates of both infested and non-infested olives were randomly collected from an area of approximately 1 ha. Each biological replicate consisted of ten drupes collected from 5 different trees. Collected olives were quickly returned to the laboratory in plastic containers and stored at 4°C for no longer than 24 h prior to lyophilization (Labconco FreeZone 2.5). Before lyophilization the fruits’ mesocarp and exocarp were separated from the stone, which was discarded. Lyophilized samples were grounded in liquid nitrogen with sterile mortars and pestles, and total DNA was extracted from 80 mg of homogenate tissues as described by Mosca and co-workers [15 (link)]. Concentration and quality of extracted DNA were assessed using Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., USA). All samples were diluted to have a DNA concentration of 20 μg·μl-1.
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8

Synthesis of Krytox-based Surfactants

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All surfactants were synthesized as described previously. 32, 39 In brief, 1 mol equivalent of Krytox FSH 157 (~6500 Da, Chemours, USA) was dissolved at 0.1 g mL--1 in Novec HFE--7100 (3M, USA).
The reaction was performed under argon using dry glassware.
The carboxylic end group of the Krytox Please do not adjust margins Please do not adjust margins and removed the top layer. This washing step was repeated three times before the surfactant was dried using a rotary evaporator (Hei--VAP, Heidolph, Germany) and a freeze dryer (FreeZone 2.5, Labconco, USA).
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9

Synthesis of Highly Conductive rGO Foams

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Materials & synthesis of rGO foams 'Highly concentrated' GO was purchased from Graphenea, as a gel at 2.5 wt%, with 495% monolayer content as measured at 0.05 wt%.
An aqueous colloidal dispersion of GO was diluted with de-ionised (DI) water into a dispersion of the desired wt%. DI water was used to introduce as few impurities and ionised particulates into the foams as possible. Impurities in the precursor dispersion, which could be introduced by using non-DI water, could impact both the mechanical and electrical properties of the resulting foam scaffolds as well as providing the potential for toxicity to the cells under study. The diluted precursor dispersion was then cast into a mould of desired geometry, frozen in a conventional freezer at approximately À18.5 1C, and lyophilised with a Labconco 'FreeZone 2.5' into a low-density foam. This mould-based liquid processing means that there is in principle no limit to the size and shape of the sample, which is beneficial when designing a scaffold for a specific application. After synthesis, the GO foams were thermally reduced into rGO for three hours at low pressure, P E 1 mBar, and set temperature T = 150 1C in a Memmert vacuum oven. This procedure is detailed schematically in Fig. 1. Reduction was undertaken until no further change in conductivity was observed and the samples had recovered a metallic lustre (Fig. S1,ESI †).
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10

Extraction of M. polymorpha Bioactives

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M. polymorpha was collected from Altay in Xinjiang Uygur Autonomous Region, China. MPEE was prepared according to our previous procedure [21 (link)]. Specifically, 100 g powders of M. polymorpha were extracted three times using 2 L of 100% ethanol. After centrifugation at 6000 rpm for 15 min, the supernatant was evaporated and freeze-dried using a Freezone 2.5 instrument (Labconco, USA). MPEE was dissolved in DMSO and the contents of flavonoids and polysaccharides were detected according to previous description [22 (link)].
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