DNA was extracted, from 0.25 g cecal content, using 700 μl Stool Transport and Recovery (STAR) buffer (Roche Diagnostics Nederland BV, the Netherlands). The cecal sample was transferred to a sterile screw-capped 2 ml tube (BIOplastics BV, the Netherlands) containing 0.5 g of zirconium beads (0.1 mm; BioSpec Products Inc., USA) and 5 glass beads (2.5 mm; BioSpec Products Inc., USA). The samples were treated in a bead beater (Precellys 24, Bertin technologies, France) at a speed of 5.5 ms− 1 for 3 × 1 min, followed by incubation at 95 °C with agitation (15 min and 300 rpm). The lysis tube was centrifuged (13,000 g for 5 min at 4 °C), and the supernatant was transferred to a 2 ml microcentrifuge tube. Thereafter, the above described process was repeated with 300 μl of STAR buffer. An aliquot (250 μL) of the combined supernatants from the sample lysis was then transferred into the custom Maxwell® 16 Tissue LEV Total RNA Purification Kit cartridge. The remainder of the extraction protocol was then carried out in the Maxwell® 16 Instrument (Promega, the Netherlands) according to the manufacturer’s instructions. DNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop® Technologies, USA), and DNA was stored at − 20 °C until further use.
Zirconium beads
Manufactured by Biospec
Sourced in Germany
Zirconium beads are a type of laboratory equipment used for various applications. They are made of zirconium, a hard and durable material. Zirconium beads are commonly used in sample preparation, homogenization, and grinding processes in scientific and research settings.
Automatically generated - may contain errors
Lab products found in correlation
Bead beater, by Biospec (3 mentions)
Mini beadbeater, by Biospec (3 mentions)
Glass bead, by Biospec (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Maxwell 16 instrument, by Promega (1 mentions)
Star buffer, by Roche (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Lightcycler probe design software, by Roche (1 mentions)
Maldi biotyper sirius system, by Bruker (1 mentions)
Magna lyser, by Roche (1 mentions)
Precellys bead beater, by Bertin Technologies (1 mentions)
Topspin 3, by Bruker (1 mentions)
600 mhz spectrometer, by Bruker (1 mentions)
Silica beads, by Biospec (1 mentions)
Lightcycler faststart dna master hybridization probes kit, by Roche (1 mentions)
Magna pure compact, by Roche (1 mentions)
Lightcycler 2, by Roche (1 mentions)
Al lysis buffer, by Qiagen (1 mentions)
24 protocols using zirconium beads
1
DNA Extraction from Cecal Contents
2
Giardia cyst isolation and quantification
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One millilitre ethanol-preserved cysts was added to 100 µl zirconium beads (Biospec products) (diameter 0.5 mm). The cysts were washed twice with 1 ml PBS by centrifugation (2 min, 2000 g ) and aspirated without disturbing the beads. PBS (170 µl) and 2 drops detection reagent (Merifluor C/G, Meridian Bioscience), containing FITC-labelled mAb against Giardia and Crytosporidium, were added to the beads–cysts suspension. The suspension was mixed gently end over end, stained for 30 min at room temperature and washed again twice as described. Finally, 200 µl PBS was added to the mixture, mixed gently and the cysts were transferred in the 200 µl (without centrifugation), while taking care not to transfer the beads. The cyst suspension was poured through a 100 µm cell strainer (Greiner Bio-One) and checked for stained Giardia cysts and the absence of Cryptosporidium with fluorescence microscopy. Cysts were counted in a modified Fuchs–Rosenthal counting chamber and adjusted to a concentration of 5 cysts (µl PBS)−1.
3
Bovine Feces DNA Extraction
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Genomic DNA was extracted using the repeated bead beating plus column method, which was previously developed for bovine feces and rumen digesta (Yu and Morrison, 2004 (link)). Briefly, the prepared sample was mixed with 0.5 g of zirconium beads (0.1 mm; Biospec products), 4 glass beads (2.5 mm; Biospec products) and 1 ml of lysis buffer (500 mM NaCl, 50 mM Tris-HCl (pH 8), 50 mM EDTA, 4 % (w/v) SDS) in 2 ml lysis tubes with screw caps (BIOplastics BV) and then processed as the published protocol. The final genomic DNA was eluted in 100 μl AE buffer (10 mM Tris-HCl, 0.5 mM EDTA; pH 9.0).
4
Optimized RNA Extraction and Gene Expression Analysis
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RNA extraction, DNase treatment and reverse transcription: The –80°C cell pellets were melted with 1 mL TRIzol reagent (Invitrogen, Cergy Pontoise, France) and broken with 0.5-mm diameter zirconium beads (Biospec products, Bartlesville, OK) in a BeadBeater (Precellys, Saint Quentin en Yvelynes, France) using two 20-s mixing sequences at a speed of 6500 rpm. RNA extraction, DNase treatment and reverse transcription were done following protocols previously described (Monnet, Back and Irlinger 2012 ).
Real-time PCR and data analyses: Oligonucleotide primers were designed using LightCycler probe design software (v1.0; Roche Applied Science, Mannheim, Germany) and synthesised by Eurogentec (Seraing, Belgium) (primers list, see Primers Table,Supporting Information ). The PCR and the data analyses were done as previously described (Monnet, Back and Irlinger 2012 ).
Real-time PCR and data analyses: Oligonucleotide primers were designed using LightCycler probe design software (v1.0; Roche Applied Science, Mannheim, Germany) and synthesised by Eurogentec (Seraing, Belgium) (primers list, see Primers Table,
5
Quantifying S. aureus in Mouse Tissues
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Samples were collected in 2 ml tubes containing 0.5 ml saline with 5 sterilized zirconium beads (Ø 2 mm, BioSpec Products) and stored on ice. To neutralize mouse endogenous antimicrobial peptide (AMP) activity, 50 μl of a 0.05% (v/v) sodium polyanethole sulfonate (SPS; Sigma) solution was added to the samples [35 (link)]. Due to technical issues, no SPS was added to samples from the P4HB, PP and sham non-infected mice at 9 days and samples from the sham infected mice at 9 days. Samples were homogenized with 3 cycles of 30 s at 7000 rpm with 30 s cooling on ice in between the cycles (MagNA Lyser, Roche). Of this homogenate, 100 μl was ten-fold serially diluted in phosphate buffered saline (PBS) and five-fold 10 μl aliquots of the sample and the dilutions were spotted on blood agar plates. After overnight incubation, colonies were counted and converted to log CFU per homogenized biopsy. Morphologically distinct colonies were selected for species determination (MALDI Biotyper, Sirius System, Bruker) to rule out contamination. In case of bacterial contamination other than S. aureus, these colonies were not included in the CFU counts.
6
Fecal Metabolic Profiling by NMR Spectroscopy
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A subset of 157 paired fecal samples (75 from Bif/Lacto group, and 81 from Control group) were analyzed by standard one-dimensional (1D) 1H NMR spectroscopy using a Bruker 600 MHz spectrometer operating at 300 K. Fecal samples were chosen for metabolic profiling pragmatically based on remaining sample quantity after previous analyses. Feces (50 mg) were combined with 700 μL of phosphate buffer (pH 7.4; 100% D2O) containing 1 mmol/L of 3-trimethylsilyl-1-[2,2,3,3-2H4] propionate (TSP), and 10 zirconium beads (1 mm diameter) (BioSpec Products). Samples were homogenized using a Precellys bead beater (Bertin) with 2 cycles of 40 s at 6,500 Hz speed, centrifuged at 14,000 g for 10 min and the supernatant was transferred to NMR tubes. 1D NMR spectra were acquired for each sample using a nuclear overhauser effect pulse sequence for water suppression as described by Beckonert and colleagues72 (link)). Spectra were automatically phased and calibrated to the TSP reference using Topspin 3.6 (Bruker BioSpin). Spectra were imported into MATLAB 9.4 (R2018a), redundant spectral regions (those arising from TSP and imperfect water suppression) were removed, and the spectral profiles were normalized using a probabilistic quotient method.
7
Real-Time PCR Assay for Pneumocystis jirovecii
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The St Paul’s Hospital laboratory-developed PJ real-time-PCR assay was adapted from one previously published (19 (link)). Briefly, 400 μL of BAL fluid in phosphate-buffered saline was extracted with 10 μL of internal amplification control according to the total nucleic acid isolation blood protocol using a Roche Magna Pure Compact instrument (Roche Molecular Diagnostics, USA) and eluted in 100 μL. Respiratory samples containing thick mucus were pretreated with proteinase K (25 μL 1 g/mL) and sodium dodecyl sulfate then lysed using zirconium beads (150 μL 2.4 mm) and silica beads (50 μL 0.1 mm) (Biospec Products, USA) before DNA extraction. Primers and fluorescence resonance evergy transfer probes were previously described, and were designed to detect a 166-base pair region of the cdc2 gene of PJ (19 (link)). PCR was performed on 5 μL of DNA extract using the LightCycler-FastStart DNA Master Hybridization Probes kit (Roche Diagnostics, Canada) in the presence of 4 mmol/L MgCl2 using the Light Cycler 2.0 (Roche Diagnostics, Canada). Melting curve analysis was performed at the end of the cdc2 PCR amplification to confirm positives. All primers and probes were synthesized by Metabion (Germany).
8
Bacterial DNA Extraction from Nasal Swabs
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Bacteria from nasopharyngeal swabs were harvested by centrifugation of 500 µL each of specimens at 30,000 g for 15 min. Each pellet was resuspended in 200 µL TE buffer (30 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 15 mg/mL lysozyme (Sigma-Aldrich, Seelze, Germany) and 20 µg/mL protease (Qiagen, Hilden, Germany) and mixed with 200 µL AL lysis buffer (Qiagen) and zirconium beads (BioSpec Products, Bartlesville, OK) prior to disruption and homogenization in a MagNa Lyser Instrument (Roche Applied Science, Germany) for 70 s at setting 7000. This was followed by 10 min incubation at 56 °C. The samples were centrifuged at 30,000 g for 5 min, and the DNA was purified using the Qiagen DNeasy blood and tissue kit according to the manufacturer’s instructions. In brief, the DNA extract was mixed with ethanol, applied to the spin column and centrifuged at 8000 g for 1 min in order to bind DNA in the lysate to the column. Then, the column was washed with Buffer AW1 (8000 g, 1 min) followed by another wash with Buffer AW2 (14,000 g, 3 min). Finally, DNA was eluted with 200 µL (2 × 100 µL) Buffer AE (8000 g, 1 min). The eluted DNA was stored at −80 °C.
9
Fecal DNA Isolation and Quantification
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DNA from fecal samples collected at the end of the intervention was isolated as previously described (32 (link)) with some minor modifications. The samples were initially mixed with 250 μL lysis buffer (Agowa, Berlin, Germany), 250 μL zirconium beads (0.1 mm), and 200 μL phenol, before being introduced to a BeadBeater (BioSpec Products, Bartlesville, OK, USA) for two × 2 min. Quantitative PCR detection was performed with the primers and according to conditions described previously (33 ). To evaluate if samples contained F19, data were plotted from low to high Ct, resulting in an S-shaped curve, with true positives in the lower and true negatives in the higher end, as previously described (33 ).
10
Fecal DNA Isolation and qPCR
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For DNA isolation, fecal samples were thawed on ice and lysed by bead beating (Mini-BeadBeater-24, Biospec Products Bartlesville, USA) for 2 min at 2800 oscillations minute− 1 in the presence of 300 μl of lysis buffer (Mag Mini DNA Isolation Kit, LGC ltd, UK), 500 μL zirconium beads (0.1 mm; BioSpec products, Bartlesville, OK, USA) and 500 μL phenol saturated with 10 mM Tris-HCl and 1 mM EDTA pH 8.0 (Sigma). After centrifugation DNA was extracted using the Mag Mini DNA Isolation Kit (LGC ltd, UK) in accordance to the manufacturers recommendations. DNA quality was assessed by routine gel electrophoresis as well as by capillary electrophoresis on the Fragment Analyzer (Advanced Analytical, Heidelberg, Germany). Quantitative PCR (qPCR) primers and probes are listed in Additional file 3 : Table S1. qPCR was performed using RT PCR master mix (Diagenode, Seraing, B) on an Applied Biosystems 7500 RT PCR system.
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