Minispin plus centrifuge

For MALDI-TOF MS spectra of I. glandulifera acid-soluble seed proteins, single seeds were macerated in 100 µl of (11 mg/ml HCCA matrix in 65% (v/v) acetonitrile, 2.5% (v/v) TFA, and 32.5% (v/v) water) (referred to as Solution 1 in the following) using the blunt end of a plastic inoculating loop. Seed debris was pelleted by centrifugation at 14,100g for 1 min in a miniSpin^® plus centrifuge (Eppendorf, Stevenage, UK). One microlitre of the resulting supernatant (or dilutions thereof in Solution 1 as indicated) was then pipetted onto the Bruker sample plate, air dried, and loaded into the spectrometer.

The thermodynamic solubility of IMA was measured for the saturated solutions in water at various pH values (pH 2: 0.01 M HCl, pH 6: 20 mM MES) buffer), pH 7.4 and 8.2: 20 mM HEPES buffer) at 25.0 ± 0.1 °C. Solutions in 10 mM concentration were attempted to make. Samples were mixed overnight, then sedimentation of the solid fraction was forced by centrifugation (Eppendorf, MiniSpin Plus centrifuge, 15 min, 12 000 rpm). The concentration of the compounds was determined by UV-vis spectrophotometry using stock solutions of the compound with a known concentration dissolved in DMSO, 50% and 10% (v/v) DMSO/buffered aqueous solution for the calibration. In the case of FAVI and MOLNU 10 mM solutions at pH 2 and 7.4 could be prepared without the presence of non-dissolved compound.

Transmission-electron microscopy (TEM) was used to determine the morphological characteristics of the isolated bacteriophage. Phage lysate in a volume of 1 mL was centrifuged at 20 °C at 14,500 rpm for 40 min (MiniSpin^® plus centrifuge, Eppendorf, Hamburg, Germany). The supernatant was removed, and the precipitate was suspended in 2 mL of 100 mM cold ammonium acetate (filtered through a 0.22 μm syringe filter). The precipitate was dissociated by repeated pipetting and centrifuged again. The whole procedure was repeated four times. After centrifugation, the precipitate was rinsed off the wall of the Eppendorf tube with 50 μL of ammonium acetate according to the procedure of Ackermann [49 (link)], with modification. Two μL of phage suspension in ammonium acetate was coated onto carbon-sputtered copper–wolfram mesh grids. After drying, the preparation was stained for 1 min in a 2% uranyl acetate solution (Warchem, Warsaw, Poland). Prepared samples were dried for 12 h at ambient temperature under sterile conditions [50 (link),51 (link)] and visualized under JEM 1400 PLUS transmission-electron microscope (Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan) at 100,000–200,000× magnification, at a voltage of 80 kV [52 (link)].

Blood samples for metabolomic analysis were taken from the vein before the morning meal. Samples (3 ml) were placed into glass tubes containing K₂EDTA (BD Vacutainer; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and centrifuged within 15 min of blood collection at 1600×g and room temperature. The resultant blood plasma was subdivided into aliquots that were pipetted into plastic tubes. These tubes were marked, transported in special thermo containers, frozen, then stored at −80°C until analysis. The analyzed samples were subjected to one freeze/thaw cycle. To test the reproducibility of this protocol, an additional set of blood samples (n = 20) were collected from the same individuals within 2–7 days of the original collection.
For plasma deproteinization, aliquots (10 µl) were mixed with 10 µl water (LiChrosolv; Merck KGaA, Darmstadt, Germany) and 80 µl methanol (Fluka, Munich, Germany) and incubated at room temperature. After 15 min, samples were centrifuged at 13000×g (MiniSpin plus centrifuge; Eppendorf AG, Hamburg, Germany) for 10 min. Deproteinized supernatants were then transferred to clean plastic Eppendorf tubes, and fifty volumes of methanol containing 0.1% formic acid (Fluka) was added to each tube. The resulting solutions were subjected to mass spectrometry analysis.

MIPs were typically prepared to 50 µl in 1.5 ml lo-bind tubes (Eppendorf #Z666505) on ice. The final MIPs contained 6% total acrylamides (w/v), of which 10% was MBAm and 90% AA. Therefore, 2.7 mg AA and 0.3 mg MBAm were combined in RS buffer, and purified human ribosomes were added to 0.75 µg/µl and gently mixed by pipetting. For control NIPs, RS buffer without ribosomes was added to AA monomers. 0.2% (w/v) APS and 0.1% (w/v) TEMED were added to the reactions, mixed and polymerized under gaseous N₂ for 10 min on ice, and for a further 10 min with the tube lid closed, resulting in gel formation. Following complete polymerisation as determined by visual inspection (ribosome/co-monomer solutions turn from colourless to semi-opaque after polymerisation), gels were manually passed through a 150 µm mesh (Endecotts Ltd) and collected. The gel fragments were washed three times with 100 µl ice cold RS buffer and collected by centrifugation at 3,500 × g for 1 min at 4 °C between washes using an Eppendorf Mini-Spin Plus centrifuge.

Metarhizium anisopliae conidial powder (2 g) was added to 20 mL chitosan solution (0.35% w/v prepared by dissolving chitosan powder in 1.5% acetic acid) followed by drop-wise addition of 12 mL sodium tripolyphosphate (0.4%). The whole mixture was then stirred for 6 h at room temperature on a magnetic stirrer (Rexim RP-1DN, Agile). The obtained homogenous slurry was added to a screw cap vial and incubated at 45 °C for 12 h. The change in color of solution from colorless to yellowish brown confirmed nanoparticle synthesis. The synthesized reaction mixture was centrifuged for 10 min at 15,000× g and 28 °C (Minispinplus centrifuge, Eppendorf, Hamburg, Germany). Pellets obtained were washed with deionized water followed by freeze drying in a refrigerator at −20 °C (BCD-576, Haier, Qingdao, China) until further use.