The quantitation of AHLs (3-oxo-C12-HSL, C4-HSL) and PQS was performed as previously described [27 –30 ]. Briefly, strains were grown at 37°C in LB broth until OD600 ≈ 1.0. For extraction of AHLs, samples were centrifuged at 10,000 g in a Thermo Fresco21 tabletop centrifuge for 20 min at 4°C. 0.6 mL of liquid supernatant was extracted with acid ethyl acetate. The organic phase was dried using a vacuum freeze dryer (CHRIST, ALPHA2-4 LD pius), resolubilized by methanol and filtered with millex (0.22 μM, MERCK). For extraction of PQS, 0.5 mL of cultures were mixed with isovolumic methanol, and samples were centrifuged at 10,000 g in a Thermo Fresco21 tabletop centrifuge for 20 min at 4°C. The supernatant was filtered with millex (0.22 μM, MERCK) and used for LC-MS/MS analysis. The following standards were used: N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL, Sigma Aldrich), N-[(3S)-Tetrahydro-2-oxo-3-furanyl] butanamide (C4-HSL, Cayman Chemical), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS, Sigma Aldrich). Three samples were performed for each group.
Millex
Manufactured by Merck Group
Sourced in United States, Germany, Ireland, United Kingdom, France, Brazil, Australia
Millex is a line of high-quality membrane filters designed for laboratory filtration applications. The core function of Millex filters is to remove particles, microorganisms, and other contaminants from liquids, ensuring sample integrity and cleanliness.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 15, by Merck Group (3 mentions)
1200 series chromatograph, by Phenomenex (3 mentions)
Microtof q 3, by Bruker (3 mentions)
Series 200, by PerkinElmer (3 mentions)
Zetasizer nano z, by Malvern Panalytical (3 mentions)
Amicon, by Merck Group (3 mentions)
Glow discharged carbon coated copper grid, by Electron Microscopy Sciences (3 mentions)
Es1000w erlangshen ccd camera, by Ametek (3 mentions)
Jem 1011 microscope, by JEOL (3 mentions)
Rezex roa organic acid column, by Phenomenex (3 mentions)
Uranyl acetate, by Ted Pella (3 mentions)
Exactive, by Thermo Fisher Scientific (2 mentions)
Accela series, by Thermo Fisher Scientific (2 mentions)
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Widepore c18 guard column, by Phenomenex (2 mentions)
0.22 m pore size nylon membrane, by Merck Group (2 mentions)
Rezex rso oligosaccharide ag 200 10 mm, by Phenomenex (2 mentions)
Itlc sg, by Pall Corporation (2 mentions)
Chromera, by PerkinElmer (2 mentions)
Series flexar, by PerkinElmer (2 mentions)
196 protocols using millex
1
Quantitative Analysis of Quorum Sensing Molecules
2
Metabolomic Profiling of Tepal Extracts
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Tepal extracts were filtered through 13 mm diameter Millex filters with 0.22 µm pore size nylon membrane (Millipore, Bedford, MA, USA). Ten microliters of filtered extract were injected using a ultra-high performance liquid chromatography device (UHPLC, Accela Series, ThermoFisher Scientific, Waltham, MA, USA) coupled to a high-resolution mass spectrometer (Exactive, ThermoFisher Scientific, Waltham, MA, USA) consisting of an Orbitrap detector and using a heated electrospray ionization (HESI) interface. Data processing was carried out through the Xcalibur software (version 4.3, ThermoFisher Scientific, Waltham, MA, USA); the XCMS metabolomics platform (Scripps Center for Metabolomics and Mass Spectrometry, La Jolla, CA, USA) and the KEGG, PUBCHEM and PHENOL-EXPLORER chemical databases, among others. For fine-tuning the analysis method, the molecular formulas of the compounds were searched in the PUBCHEM platform and entered in the Qual Browser package of the Xcalibur software, where mass/charge (m/z) ratios of each metabolite were identified in the negative mode, adjusting a mass tolerance of ≤2 ppm in the Processing Setup Package. Additionally, correlations between compounds of the same metabolic pathway and the LogP coefficient were used to accurately identify these metabolites.
3
Neuronal Cocultures for Antibody Evaluation
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Primary rat hippocampal neuron and astrocyte cocultures were prepared from embryonic day 18 to 19 as previously described.17 (link),18 (link) Patient or control CSF was collected and filtered using Millex filters (Millipore, Billerica, MA). High-titer CSF was diluted 1:20–100 to treat neurons in vitro for 24 hours or as stated. Immunoglobulin G (IgG) from the serum of 1 patient (02066) was collected and filtered using protein A/G Sepharose columns as described.18 (link) Treatment with patient IgG (∼20μg/ml) or serum (1:200 dilution) decreased synaptic AMPAR clusters to a similar extent as treatment with CSF (see Results), without side effects to culture health. Patient CSF was used to treat neurons unless otherwise stated. In surface biotinylation experiments, control or patient sera were used to treat neurons (1:200 dilution). Each CSF was tested for antibody reactivity by staining mouse or rat brain sections and human embryonic kidney (HEK) cells expressing GluA1/GluA2 heteromers of the AMPAR as previously described.5 (link)
4
Monitoring npGG Particle Stability
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To determine the stability of the produced GG particles over time, a 1 mg.mL−1 suspension of npGG was prepared in different solutions (culture medium, phosphate buffer saline (PBS), and deionized water) and filtered through a 0.8 µm filter (Millex, Millipore, France). Particle size and dispersity was evaluated over 7 days by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK). Between data acquisition times, samples were stored at room temperature.
5
Bacterial Surface Shaving Protocol
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The H. pylori strain J99 was grown on WC dent agar plates. Then, bacteria were harvested by scraping and split with regard to unfixed and PFA fixed conditions. Subsequently, bacteria were washed twice with PBS and resuspended in shaving buffer (1x PBS, 20% (w/v) sucrose, 10 mM DTT) to a final OD600 30. 1 ml of the bacterial solution was distributed into 1.5 ml reaction tubes on ice. 10 µg sequencing grade Trypsin was added to one half, and the trypsin’s formulation buffer (50 mM acetic acid) added to the other half. Subsequently, bacteria were incubated at 37 °C under shaking in a Thermomixer (Eppendorf) at 500 rpm. After 10, 20 and 30 minutes, shaving was stopped by placing the corresponding tube on ice. Bacteria were pelleted by centrifugation and the supernatant filtered through a 0.22 µM syringe filter (sterilized by gamma irradiation, Millex®, Millipore). The supernatant was shock frozen in liquid nitrogen and stored at −20 °C until preparing the samples for MS analysis.
After each step viability was measured by counting CFU in triplicates and at the end morphology analyzed by microscopy. The experiment was performed independently in biological quadruplicates.
After each step viability was measured by counting CFU in triplicates and at the end morphology analyzed by microscopy. The experiment was performed independently in biological quadruplicates.
6
Isolation and Purification of Bacterial OMVs
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OMVs were isolated as previously described (21 (link)). Briefly, 50 ml of each bacterial culture was normalized to an OD595 of 0.5 and centrifuged (12,000 × g, 4°C, 20 min). Cultures were grown for 48 h in order to obtain a robust and quantifiable amount of OMVs. Supernatants were harvested and sequentially filtered through 0.8-μm and 0.45-μm membrane filters (Millex, Millipore). The resulting filtrates were concentrated using a 10-kDa molecular weight cutoff centrifugal filter unit (Amicon Ultra-15, 2,360 × g, 4°C, 20 min; Millipore). Concentrated filtrates were then centrifuged (200,000 × g, 4°C, 2.5 h) in a tabletop Optima ultracentrifuge (Beckman Coulter). Pelleted OMVs were resuspended in 300 μl of phosphate-buffered saline (PBS).
7
Phage Induction and Transduction Assay
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The supernatant of stationary-phase 300-169 cells with/without MMC (1 μg/ml; Sigma; as an agent to induce the liberation of temperate phages) (41 (link)) exposure and plasma from 300-169 strain-infected rabbits in an experimental infective endocarditis (IE) model (see detailed description under “Experimental IE model” below) were filtered (0.22 μm; Millex; Millipore Corp.) to remove bacterial cells, diluted in phage buffer (42 (link)), and mixed with a recipient strain, RN4220 (a well-studied prophage-free reference S. aureus strain). The mixtures were plated on TSA plates using a well-established double-layer technique (42 (link), 43 (link)) and incubated at 37°C overnight or until plaques developed.
8
HPLC Analysis of Sugars
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Each extract was filtered through 0.45 μm nylon filters (Millex, Millipore, Bedford, VA). The filtrate (20 μL) was injected into the HPLC system mentioned above. The analyses were carried out on an Agilent Hi-Plex Ca column (8% crosslinked, 7.7 × 300 mm, 8 μm) using MilliQ water as the mobile phase in an isocratic mode. The flow rate was 0.6 mL min−1 and the column temperature was set at 85 °C. Results were expressed as ppm sugar determined g−1 of dry weight.
9
Purification of Amylase Enzyme
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Extracellular proteins were precipitated with ammonium sulfate (to the final saturation of 80 %, at 4 °C) for several hours. After centrifugation (4234×g, 4 °C, 45 min), the proteins were resuspended in binding buffer (phosphate buffer, 20 mM, pH 7.4; NaCl, 0.5 M; imidazole, 20 mM), filtered through a 0.45-μm syringe filter (Millex, Millipore), and loaded onto the ÄKTA FPLC system (ÄKTA Pharmacia GE FPLC) equipped with a HisTrap HP column (5 mL, GE Healthcare), with Ni2+ ions immobilized on sepharose. The purification procedure was carried out under increasing gradient of elution buffer (phosphate buffer, 20 mM, pH 7.4; NaCl, 0.5 M; imidazole, 0.5 M) at the flow rate of 5 mL/min. Fractions were immediately analyzed for the amylase activity and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), to identify the enzyme-enriched fraction and assess its purity.
10
LAB Strain Cultivation and Extraction
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The LAB strains were cultivated in MRS broth at 37°C for 16 h∼18 h. The fermentation broth was centrifuged at 8000 rpm for 5 min under 4°C and the supernatants were collected and filtered through a Millex® 0.22-μm filter (Millipore) to obtain cell-free supernatant (CFS). The harvested cell pellets were washed twice with 0.1 mM phosphate buffer solution (PBS, pH 7.4), re-suspended in PBS, and adjusted to 109 CFU/mL. The re-suspended solutions were divided into two aliquots. One aliquot was used as intact cells (IC), and the other aliquot was ultra-sonicated at 800 W for 30 min (5 s sonication, 7 s interval) in an ice bath. The resulting supernatant was harvested and was filter sterilized to obtain intra-cellular cell-free extracts (CFE).
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