Fastprep 24 5g instrument

M. bovis BCG wild type or mutant cultures were grown to mid-log phase in independent biological replicates and 100 mL of culture (adjusted to OD₆₀₀ = 0.4) was harvested by centrifugation at 3400 × g for 20 min at 277 K, washed with ice-cold phosphate-buffered saline and pelleted again. For the western blotting experiments, mid-log cultures were adjusted to OD₆₀₀ = 0.2 in fresh media and subjected to POA treatment (1 or 4 mM), the equivalent of 100 mL OD₆₀₀ = 0.4 was harvested at specified time points. The cell pellets were resuspended in 600 µL lysis buffer (50 mM Tris/Hcl pH 7.5, 5% (vol/vol) glycerol, 1.5 mM MgCl₂, 150 mM NaCl, 1 mM DTT, 1% n-dodecyl β-D-maltoside (w/vol), 1× complete EDTA-free protease inhibitor cocktail (Roche)), transferred to a lysis matrix B tube (MP Biomedicals) and homogenized using a FastPrep-24 5 G instrument (MP Biomedicals). The cell debris was pelleted by centrifugation at 13,000 × rpm for 10 min at 277 K and the supernatant (about 400 µL) was collected and stored at 193 K until further use. The protein concentration was determined by a BCA protein assay kit (Pierce).

The extraction of RNA was performed with the same method for both the microarray and PCR. The RNA was isolated with the Nucleospin miRNA isolation kit (Machery-Nagel, Düren, Germany), following the manufactures instructions for extraction of total RNA. The artery-samples were first homogenized in in Lysing matrix D tubes containing 1.4 mm ceramic spheres (MP Biomedicals, CA, USA) and lysis buffer (ML buffer) from the NucleoSpin kit on dry ice in a FastPrep-24™ 5G instrument (MP Biomedicals, USA) with 3x20sec cycles.
After RNA extraction, the amount of RNA was quantified using a NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scientific, MA, USA). A ratio of sample absorbance at 260 nm and 280 nm in the range of 1.7 to 2.1 was accepted. The RNA which was to be used for microarray analysis was concentrated with a Scan Speed 32 speed vacuum concentrator (Labogene, Denmark). The concentration and quality of the concentrated RNA was determined with a NanoDrop ND1000 spectrophotometer (ThermoFisher Scientific, MA, USA).

The thermal stabilities of the encapsulated bioactive compounds were evaluated using a thermostatic water bath at 90 °C for up to 60 min. A 1 mL suspension of the cells with encapsulated compounds and 1 mL of juice alone were added to the prewarmed 20 mL glass vials (Thermo Scientific™ B780020, Waltham, MA, USA) and incubated in the dark for 1, 2, 5, 10, 20, 40, and 60 min. The concentration of the total antioxidant contents in the juice sample and the cell encapsulated sample were maintained the same. After the treatment, 1 mL acidified methanol was added to each vial. Bead-beating at 6.0 m/s for 30 s for 3 times (FastPrep-24™ 5G Instrument, MP Biomedicals, Irvine, CA, USA) was then carried out to facilitate thorough extraction. Finally, the homogenized samples were sonicated using a bath sonication device (Branson 2510 Ultrasonic Cleaner, Branson Ultrasonics Corp., Danbury, CT, USA) for 10 min. The methanolic extract was then centrifuged to remove cell debris and the supernatant was used for subsequent anthocyanin measurement and total antioxidant capacity quantification as described in Section 3.6 and Section 3.7

The viability of the slices was determined by measuring ATP levels [39 (link)]. The slices were collected separately; each slice was put into 1 mL of ethanol solution (70% (v/v) containing 2 mM ethylenediaminetetraacetic acid, pH 10.9), immediately frozen and stored at −80 °C until further analyses. After thawing, the slices were homogenized with the FastPrep-24 5G Instrument (MP Biomedicals, Santa Ana, CA, USA) and centrifuged for 5 min at 12,000× g at 4 °C. ATP content was measured in supernatant using the ATP Bioluminescence Assay Kit CLS II (Roche, Mannheim, Germany) in a black 96-well plate according to the manufacturer's protocol using plate reader Tecan Infinite M200 (Tecan Group, Männedorf, Switzerland) and a standard ATP calibration curve. The concentration of ATP was corrected for the total protein content from the remaining sample pellet. The sample pellet was dissolved in 200 μL of 5 M NaOH for 30 min at 37 °C after dilution by water to 1 M NaOH. The protein content was estimated using the BCA assay kit, using bovine serum albumin for the calibration curve.

Nematode-associated bacteria were identified by comparing the bacterial communities of H. axyridis beetles from three different rearing groups: nematode-free greenhouse-reared, laboratory-reared, and nematode-infected greenhouse-reared beetles. For each rearing group, three beetles were surface sterilised with 80% ethanol and 0.3% bleach, and dissected individually in sterile PBS. Body fluids and/or nematodes in PBS were transferred to individual reaction tubes and centrifuged briefly before homogenization with three 2.3-mm zirconium/glass beads (BioSpec Products) in a FastPrep-24™ 5G instrument (MP Biomedical) at 10 m/s for two periods of 45 s. We then transferred 100-µl extracts onto lysogeny broth agar plates and incubated them at 37 °C for 2 days. We amplified the 16S SSU rRNA gene from a total of 90 colonies (ten colonies per beetle sample) using the universal 5′ primer 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and universal 3′ primer 1492R (5′-CGG TTA CCT TGT TAC GAC TT-3′). The positive PCR products (from 73 colonies) were treated with Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (Sigma-Aldrich) for enzymatic removal of excess nucleotides and primers prior to direct sequencing and the obtained sequences were analysed as described above.

All fresh frozen tissues were placed in lysis buffer with lysing matrix Z (MP Biomedicals, Santa Ana, CA, USA) and homogenized using the FastPrep-24™ 5G Instrument (MP Biomedicals, Santa Ana, CA, USA). The RNA was then extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. RNA integrity was assessed using the RNA Nano 6000 Assay Kit and the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).

In a 2-ml screw cap tube containing 500 mg of 0.1-mm silica spheres (MP Biomedical, Solon, OH, USA), 200 mg of cecal content, and 700 μl of lysis buffer [Tris-HCl 500 mM pH 8, EDTA 100 mM pH 8, NaCl 100 mM, SDS 1% (w/v)] were mixed together. A 900-μl volume of lysis buffer was used as a negative control. A mechanical lysis step was performed using a FastPrep-24™ 5G Instrument (MP Biomedical) for three runs of 60 s each, at 6 m/s. Samples were kept on ice during 5 min between each run. A second step involving thermal lysis was carried out on the samples that were heated for 20 min at 95°C and kept for 5 min on ice at the end of the procedure. The supernatant was collected after a centrifugation at 18,000 × g for 15 min and a standard phenol/chloroform purification protocol was used to complete the DNA extraction (11 (link)). The DNA concentration of each sample was measured using a QFX Fluorometer (Froggabio, Toronto, ON), and the purity of those samples was assessed using a Nanodrop 1000 (Fisher, Ottawa, ON) device. DNA samples were stored at −20°C until analysis.