Pristine soil (sieved using a 4 mm sieve) was contaminated with diesel at 6.4% (v/w), then mixed and left in the fume hood for 24 h. The soil was mixed, and 180 g was dispensed into 30 glass containers, then placed inside a plastic mesocosm of equal diameter. The appropriate treatments were added to different mesocosms in triplicate, as described in Table 1 . For the bacteria only treatment, bacteria (45 mL) were centrifuged (Heraeus Megafuge 8R, Thermo Scientific, Waltham, MA, USA) and resuspended in 5 mL of 0.9% NaCl. Sodium chloride (5 mL, 0.9%) was added to the mesocosms for all other treatments. Water (1.5–4.2 mL) was added at least once every two weeks for the first five (5) weeks. From week 6, the moisture content was regulated to 11–18% once every week by adding water when necessary. The moisture content was measured by drying the soil sample in an oven for at least 12 h at 105 °C. The weight of the pot was measured weekly, and water was added when necessary to achieve the required moisture. The soil was mixed at least once every week and sampled in weeks 3, 6, 10, 14, 18 and 22.
Heraeus megafuge 8r
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
The Heraeus Megafuge 8R is a high-performance refrigerated centrifuge designed for a wide range of laboratory applications. It features a robust and efficient design to provide reliable and consistent results.
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Lab products found in correlation
Specord 200 plus, by Analytik Jena (1 mentions)
Polycarbonate membrane filter, by Cytiva (1 mentions)
Qev column, by Izon Science (1 mentions)
Mini extruder, by Avanti Polar Lipids (1 mentions)
Victor3 multilabel counter, by PerkinElmer (1 mentions)
Beh c18 2.1 50 mm 1 7 μm column, by Waters Corporation (1 mentions)
1290 lc system, by Waters Corporation (1 mentions)
Qtrap 6500, by AB Sciex (1 mentions)
8 protocols using heraeus megafuge 8r
1
Soil Bioremediation in Mesocosms
2
Synthesis of TiO2 Nanomaterials via Hydrothermal Method
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Commercial TiO2 (Aeroxide P25, 1.2 g) and 75 mL of a 10 M NaOH aqueous solution were placed in a 95 mL teflon lined hydrothermal autoclave reactor. After stirring and ultrasound treatment for 1 h, the reactor was kept in the oven at 110°C for 12 h. Next, the sample was poured into six 50 mL polypropylene conical tubes (BD Falcon), washed with DI water and separated from the supernatant through a centrifuge (at 8000 rpm, model Heraeus Megafuge 8R, from Thermo Scientific) several times, until the conductivity of the supernatant was below 70 μS/cm. Afterwards, the 6 tubes containing the powder were filled with a 0.1 M HCl aqueous solution, sonicated for 15 min and centrifuged once. Afterwards, the samples were washed with DI water (as described above) till the conductivity of the supernatant was around 2 μS/cm. The conductivity was measured by a Delta Ohm pH-χ-O2 meter (model HD 22569.2) and SPT 01 G probe (from Delta Ohm). The obtained powders were kept in oven at 80°C for 12 h and, eventually, grinded.
3
Bradford Protein Quantification Method
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The amount of protein was determined by using the Bradford method [58 ]. Concisely, after sonication/conventional extraction, each sample was centrifuged (Thermo Scientific Heraeus Megafuge 8R-Germany) at 4 °C at 7508× g for 20 min, and the supernatant was collected and diluted. Then, 1 mL from each diluted solution was mixed with 5 mL of Coomassie brilliant blue G-250 solution, and the mixture was kept for 2 min at room temperature. The absorbance of each mixture was taken at 525 nm by a UV-Vis Double-Beam spectrophotometer (Specord-200 Plus, Analytik Jena, Germany). Bovine serum albumin (0–100 ppm) was used to prepare a standard curve for quantifying the protein contents of the extracts. Based on the protein content of the extract, the protein extraction yield (g/100 g sample) was calculated using following formula:
4
HPLC Analysis of PK11195 in Tissues
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On the day of the HPLC experiment, ultrapure water was added to the defrosted tissue samples (heart, lungs and spleen 1:1, w/v; and brain 1:3, w/v) in order to facilitate homogenization. The homogenized tissues and arterial blood samples were centrifuged at 2000 rpm 4 °C (24 position rotor, Heraeus Megafuge 8R, Thermo Scientific) for 5 min. The whole blood and tissue supernatants were collected and centrifugation was repeated 3 times under the same conditions in order to recover as much whole blood and tissue supernatant as possible. Freezing-thawing cycle of the whole blood sample causes cell lysis, therefore the resultant supernatant represents the whole blood drug content, in line with previously described blood sample collection and storage/processing methods [18 ,19 (link)]. The cell-free tissue and whole blood and tissue supernatants were denatured using acetonitrile (1:1.4, v/v) and centrifuged at 2000 rpm 4 °C for 4 min. Following protein precipitation, the final supernatants were collected for HPLC analysis.
The prepared samples were analysed using the method described inSection 2.3.1 . The measured PK11195 peak area was converted to concentration by applying linear regression equation obtained from the calibration curve.
The prepared samples were analysed using the method described in
5
Isolation and Purification of Extracellular Vesicles
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U937 or anti-PSMA transfected U937 cells were washed three times with PBS, centrifuged for 5 min at 1500 rpm (Heraeus Megafuge 8R, Thermo Scientific, UK), and resuspended in PBS at a concentration of 5x10 6 cells/ mL. Cell suspensions were sequentially extruded through 10 µm (10x); 5 µm (10x); 1 µm (10x); 0.4 µm (10x); 0.2 µm (5x) and 0.1 µm (5x) polycarbonate membrane filters (Whatman) using the mini-extruder (Avanti Polar Lipids, AL USA). To purify EMs from soluble proteins, 500 µL of extruded EMs sample was loaded to a qEV column (Izon Science). As the EMs sample entered the column top filter, more PBS was added, and 500 µL fractions were collected. The first six fractions (3 mL) are considered as the void volume and did not contain any EMs. Fractions 7, 8, 9 and 10 which contain pure EMs were collected and concentrated using Amicon Ultra-4 10k MWCO tubes (Sigma Aldrich, UK). PSMA-EMs-N and PSMA-EMs-L refer to EMs prepared from PSMApeptide expressing U937 cells engineered using nucleofection and lentiviral transduction, respectively.
6
Lentiviral Transduction of THP-1 Cells
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If not stated otherwise, all media and supplements were purchased from Thermo Fisher Scientific. The human THP-1 cell line from monocytic origin was grown in full growth medium of RPMI1640 medium supplemented with 10% FCS, 50 µg/ml Gentamycin and GlutaMax. Cells were maintained between 0.5 and 1 Mio cells/ml by addition or replacement of complete growth medium. Cells were cultured in 10 cm or 15 cm petri dishes (greiner) in 10 ml or 20 ml culture medium, respectively. Cells were monitored with a light microscope (IT40 5PH, VWR) and grown under controlled conditions at 37°C and 5% CO2. For cell splitting, cells were collected and centrifuged at 500g for 3 min (Heraeus Megafuge 8R, Thermo Fisher Scientific). The supernatant was aspirated and cells were resuspended in fresh growth medium. Cell lines expressing Langerin were generated as described before1. Briefly, human Langerin cDNAs (Sinobiologicals) were cloned into a lentiviral BIC-PGK-Zeo-T2a-mAmetrine : EF1A construct by Gibson assembly (NEB) according to the manufacturer’s protocol. Hek293 cells were transfected S4 with the lentiviral vector together with third-generation packaging vectors and viral particles were then used for transduction of THP-1 cell lines.
7
Formulation and Characterization of Chitosan Nanoparticles
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Chitosan Nanoparticles (CSNP)
CSNP were formulated via the ionic gelation method. 27 Briefly, chitosan was dissolved in 1% acetic acid at a concentration of 1 mg/ mL and pH of 5.5. A TPP solution (0.45 mg/mL) containing 25 mg/mL FITC-OVA was prepared in PBS. To prepare CSNP, 5 mL of the FITC-OVA containing TPP solution was added dropwise to 5 mL of the chitosan solution and allowed to stir for 12 min, followed by centrifugation at 8000 g (Heraeus Megafuge 8R, Thermo Fisher Scientific, Waltham MA) for 20 min. The supernatant was separated and centrifuged for a further 20 min at 8000 g for maximal particle collection. To calculate the loaded amount of FITC-OVA, the supernatant was further centrifuged (28,000 g, 60 min) to ensure sedimentation of all suspended particles. The supernatant was collected and analysed for fluorescence (excitation 485 nm, emission 535 nm, Victor 3 Multilabel counter, PerkinElmer, Waltham MA) and compared to a FITC-OVA standard curve to quantify the amount of unencapsulated FITC-OVA. Loaded amount of FITC-OVA was calculated as percentage relative to chitosan mass.
CSNP were formulated via the ionic gelation method. 27 Briefly, chitosan was dissolved in 1% acetic acid at a concentration of 1 mg/ mL and pH of 5.5. A TPP solution (0.45 mg/mL) containing 25 mg/mL FITC-OVA was prepared in PBS. To prepare CSNP, 5 mL of the FITC-OVA containing TPP solution was added dropwise to 5 mL of the chitosan solution and allowed to stir for 12 min, followed by centrifugation at 8000 g (Heraeus Megafuge 8R, Thermo Fisher Scientific, Waltham MA) for 20 min. The supernatant was separated and centrifuged for a further 20 min at 8000 g for maximal particle collection. To calculate the loaded amount of FITC-OVA, the supernatant was further centrifuged (28,000 g, 60 min) to ensure sedimentation of all suspended particles. The supernatant was collected and analysed for fluorescence (excitation 485 nm, emission 535 nm, Victor 3 Multilabel counter, PerkinElmer, Waltham MA) and compared to a FITC-OVA standard curve to quantify the amount of unencapsulated FITC-OVA. Loaded amount of FITC-OVA was calculated as percentage relative to chitosan mass.
8
Investigating Fenofibrate Release from MMC
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A shift in the UV spectrum occurred during the release of fenofibrate-loaded MMC, so UPLC-MS was used to determine the origin of the shift. Two samples were weighed into Eppendorf tubes, one with 8 mg fenofibrate-loaded MMC and the other with 0.7 mg crystalline fenofibrate and 7.2 mg MMC. Two milliliters of Milli-Q water was added to the tubes, which were then placed on a plate shaker at 37 °C for 24 h. The supernatants were recovered by centrifugation at 37 °C for 15 min at 23,000×g (Heraeus Megafuge 8R, ThermoScientific) and analyzed by obtaining a full MS spectrum (QTRAP 6500, AB Sciex). A stock solution of fenofibrate dissolved in DMSO was used as reference. Separation was performed on an Agilent 1290 LC-system, with a Waters BEH C18 2.1 × 50 mm (1.7 μm) column, and the run was made in positive mode. The mobile phase consisted of (A) 5% acetonitrile, 0.1% formic acid, and 94.9% Milli-Q water; and (B) 95% acetonitrile, 0.1% formic acid, and 4.9% Milli-Q water. A constant flow rate of 0.5 mL/ min was used for the gradient elution as follows: A was constant at 99% for 1.0 min, then decreased linearly to 20% for 7.30 min. A was thereafter decreased to 10% for 0.2 min, kept constant for 0.5 min, and then increased to 99% for 0.5 min.
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