Gramicidin S hydrochloride (Sigma-Aldrich; 1 mM) was prepared by dissolving commercial GS in 10 mL sterile distilled water and passing the solution through a 0.22 μm membrane filter (Millipore, UK). This stock solution was used to prepare the following concentrations of GS: 10, 30, 50, 100, 300, 500 and 700 μM. One milliliter of each concentration was submitted to LC-MS analysis (see below) and peak areas obtained by the total ion chromatogram (TIC) calculated. Samples were analyzed in triplicate, and a calibration curve prepared.
0.22 μm membrane filter
Manufactured by Merck Group
Sourced in United States, Germany, Ireland, United Kingdom
The 0.22 μm membrane filter is a laboratory equipment designed to remove particulates and microorganisms from liquids. It has a pore size of 0.22 micrometers, which allows the passage of smaller molecules while effectively trapping larger particles and microbes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Sea salt, by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Sep pak c18, by Waters Corporation (2 mentions)
Prominence lc solution system, by Shimadzu (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ht7700, by Hitachi (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Xpn 100, by Beckman Coulter (2 mentions)
Mrs medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Euroclone (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Microaerobic chamber, by Don Whitley Scientific (1 mentions)
Columbia agar base, by Thermo Fisher Scientific (1 mentions)
Campylobacter selective supplement, by Thermo Fisher Scientific (1 mentions)
Brucella broth, by Thermo Fisher Scientific (1 mentions)
Sonifier 450, by Emerson (1 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (1 mentions)
137 protocols using 0.22 μm membrane filter
1
Gramicidin S Quantification by LC-MS
2
Campylobacter jejuni Virulence Factor Extraction
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C. jejuni ATCC 33291 and C. jejuni 11168H cdtA mutants were grown at 37b °C in a microaerobic chamber (Don Whitley Scientific, Shipley, United Kingdom) containing 85% N2, 10% CO2, and 5% O2, either on blood agar (BA) plates containing Columbia agar base (Oxoid, Basingstoke, United Kingdom) supplemented with 7% (v/v) horse blood (TCS Microbiology, United Kingdom) and Campylobacter Selective Supplement (Oxoid), or in Brucella broth (Oxoid) with shaking at 75 rpm. C. jejuni strains were grown on BA plates for 24 h prior to use in all assays, unless otherwise stated.
C. jejuni strains were grown in 50 mL Brucella broth (Oxoid) at 37 °C in a shaking incubator under microaerophilic condition for 48 h. The bacterial suspensions were centrifuged at 4000 rpm for 10 min and the pellets were resuspended in 20 mL of Dulbecco’s modified eagle medium (D-MEM) (Sigma-Aldrich, St Louis, MO, USA). Then, bacterial suspensions were adjusted spectrophotometrically to approximately 108 bacteria/mL and lysed by sonication (2 × 30 s bursts with 30 s intervals between each burst) by using a sonicator (Sonifier 450, Branson, Danbury, CT, USA). Cell debris and un-lysed bacterial cells were then removed by centrifugation at 4000 rpm for 10 min. Aliquots of each lysate were sterilized by a 0.22-μm membrane filter (Millipore, Milano, Italy) and stored at −20 °C before use [68 (link)].
C. jejuni strains were grown in 50 mL Brucella broth (Oxoid) at 37 °C in a shaking incubator under microaerophilic condition for 48 h. The bacterial suspensions were centrifuged at 4000 rpm for 10 min and the pellets were resuspended in 20 mL of Dulbecco’s modified eagle medium (D-MEM) (Sigma-Aldrich, St Louis, MO, USA). Then, bacterial suspensions were adjusted spectrophotometrically to approximately 108 bacteria/mL and lysed by sonication (2 × 30 s bursts with 30 s intervals between each burst) by using a sonicator (Sonifier 450, Branson, Danbury, CT, USA). Cell debris and un-lysed bacterial cells were then removed by centrifugation at 4000 rpm for 10 min. Aliquots of each lysate were sterilized by a 0.22-μm membrane filter (Millipore, Milano, Italy) and stored at −20 °C before use [68 (link)].
3
Isolation and Propagation of S. ureilyticus Phage
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To isolate the phage infecting S. ureilyticus, environmental water samples were collected from a sewage treatment plant in Daejeon (Republic of Korea) using S. ureilyticus strain ATCC 49330. Phage isolation and propagation were performed using the double-layer agar method as previously described (Melo et al., 2014 (link); Kim et al., 2019 (link)). Briefly, 1 ml of S. ureilyticus suspension was inoculated into the mixture of collected wastewater and TSB. Following overnight incubation at 37°C, the mixture was centrifuged at 10,000 × g for 20 min to remove the bacterial pellet, and the supernatant was filtered through a 0.22-μm membrane filter (Millipore, USA) to secure phages from the mixture. The filtrate was used to detect the presence of phages by confirming the formation of plaques on bacterial lawns. Following overnight incubation, plaque picking was repeated at least three times until a single plaque morphology was observed. For phage propagation, isolated phage suspension was incubated overnight with host bacteria by double-layer agar method, and the top-agar layer was collected, centrifuged at 10,000 × g for 10 min, and filtered. The filtered phage lysates were stored with 15% glycerol at −80°C.
4
Preparation of Marine Culture Media
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Three culture media (ASW, ASW-LB, and ASW-M9) were used in this study. ASW was made of 40 g/L sea salt (Sigma, USA) and sterilized by passing through a 0.22 μm membrane filter (Millipore, USA). ASW-LB was prepared by mixing ASW with 10× of LB (9:1, v/v) to make a final concentration of 1% tryptone and 0.5% yeast extract. ASW-M9 was prepared by mixing ASW with 10× of M9 (9:1, v/v) to obtain a final concentration of 0.4% glucose as the carbon source and 0.1% NH4Cl as the nitrogen source.
5
Preparation of HICA Stock Solution
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A 100 mg/mL stock solution of HICA was prepared by dissolving DL-HICA (Sigma-Aldrich, USA) in sterile Milli Q water (Milli-Q®, Germany) and filter sterilised using a 0.22 μm membrane filter (Millipore, Ireland). The solution was further diluted to different concentrations as required in each assay.
6
Saliva Collection and Preparation for Downstream Analysis
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Human saliva samples from 3 healthy adult male volunteers were collected on ice, with the approval of the Ethics Committee for Research in Humans (CAAE 26142014.0.0000.5416) and after informed consent was obtained. None of the participants had been treated for oral diseases or had taken any prescription medication during the 3 months before the study [29 (link)]. For standardization, all saliva was collected at the same time of day. The saliva was prepared as described in previous studies [30 (link)]. Before its use, the supernatant obtained after centrifugation at 45 N for 15 minutes at 4°C was purified with a 0.22 μm membrane filter (Millipore, Burlington, MA, USA) and stored at −80°C [31 (link)32 (link)].
7
Optimization of Tumor-Associated Macrophage Induction from RAW264.7 Cells
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The mouse macrophage cell line RAW264.7 (M0) and Lewis lung carcinoma (LLC) cell line (Qilu College of Medicine, Shandong University, China) were grown in RPMI-1640 with 10% fetal bovine serum (Gibco BRL Life Technologies, Paisley, UK) at 37°C, under 5% CO2 atmosphere.
RAW264.7 cells were cultured in folate-deficient RPMI 1640 (FDRPMI) medium containing 10% fetal bovine serum (FBS) to induce folate receptor (FR) up-regulation.20 The expression fold changes of Folr were identified by qPCR. The use of all cell lines was approved by the Research Ethics Committee of Qilu Hospital of Shandong University.
TAMs were prepared by culturing RAW264.7 cells (M0) in the conditioned medium (CM) according to previous study.21 (link) The CM was the supernatants of LLCs which were harvested and filtered through a 0.22 μm membrane filter (Millipore, NY, USA). To verify the activation of M0 macrophages to TAMs, optical microscopy was used to describe morphological changes, and qPCR was applied to determine the up-regulated expressions of Mrc1, Il0, Nos2, and Tnf. Different concentrations (0, 50%, 100%) of CM were used to culture M0 cells at different time points (0, 24, 48, 72 hrs) to optimize macrophage activation.
RAW264.7 cells were cultured in folate-deficient RPMI 1640 (FDRPMI) medium containing 10% fetal bovine serum (FBS) to induce folate receptor (FR) up-regulation.20 The expression fold changes of Folr were identified by qPCR. The use of all cell lines was approved by the Research Ethics Committee of Qilu Hospital of Shandong University.
TAMs were prepared by culturing RAW264.7 cells (M0) in the conditioned medium (CM) according to previous study.21 (link) The CM was the supernatants of LLCs which were harvested and filtered through a 0.22 μm membrane filter (Millipore, NY, USA). To verify the activation of M0 macrophages to TAMs, optical microscopy was used to describe morphological changes, and qPCR was applied to determine the up-regulated expressions of Mrc1, Il0, Nos2, and Tnf. Different concentrations (0, 50%, 100%) of CM were used to culture M0 cells at different time points (0, 24, 48, 72 hrs) to optimize macrophage activation.
8
Metagenomic analysis of microbial communities
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The DNA
extraction and Miseq were performed according to Guo et al.63 (link) Briefly, the microorganisms in cultivation were
collected with a 0.22 μm membrane filter (Millipore). DNA was
extracted using the UltraClean Soil DNA Isolation Kit (Mobio). The
DNA was extracted and quantified. The archaea-specific primer pairs
Arch344F/Arch915R30 (link),67 ,68 (link) and fungi-specific primer pairs ITS1f/ITS2R35 (link) were used to amplify the archaeal 16S rRNA gene and the fungal ITS
gene, respectively. The PCR was performed as follows: 95 °C for
5 min, 27 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C
for 45 s, and finally 72 °C for 10 min. Sequencing was performed
using an Illumina Miseq platform.
All of the sequence reads
were quantified and primer dimers were removed.69 (link) Operational taxonomic units (OTUs) were assigned at 97%
similarity using Usearch v 7.1. The 16S rRNA gene sequences were compared
against the Silva database,70 (link) and the fungal
gene sequence was compared against the Unite fungal database.71 (link) Diversity and richness estimators were calculated
by Mothur v.1.30.1.72 (link) The sequences derived
from Miseq have been deposited in the NCBI Sequence Read Archive with
accession number PRJNA657731.
extraction and Miseq were performed according to Guo et al.63 (link) Briefly, the microorganisms in cultivation were
collected with a 0.22 μm membrane filter (Millipore). DNA was
extracted using the UltraClean Soil DNA Isolation Kit (Mobio). The
DNA was extracted and quantified. The archaea-specific primer pairs
Arch344F/Arch915R30 (link),67 ,68 (link) and fungi-specific primer pairs ITS1f/ITS2R35 (link) were used to amplify the archaeal 16S rRNA gene and the fungal ITS
gene, respectively. The PCR was performed as follows: 95 °C for
5 min, 27 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C
for 45 s, and finally 72 °C for 10 min. Sequencing was performed
using an Illumina Miseq platform.
All of the sequence reads
were quantified and primer dimers were removed.69 (link) Operational taxonomic units (OTUs) were assigned at 97%
similarity using Usearch v 7.1. The 16S rRNA gene sequences were compared
against the Silva database,70 (link) and the fungal
gene sequence was compared against the Unite fungal database.71 (link) Diversity and richness estimators were calculated
by Mothur v.1.30.1.72 (link) The sequences derived
from Miseq have been deposited in the NCBI Sequence Read Archive with
accession number PRJNA657731.
9
Quantification of Free Amino Acids in Sprouts
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Free amino acids were analyzed following the procedure of Je et al. [55 (link)] with some modifications. One gram of sprout sample was hydrolyzed with 6 N HCl (10 mL) in a sealed-vacuum ampoule at 110 °C for 24 h. The HCl was removed from the hydrolyzed sample on a rotary evaporator, the content was mixed with 0.2 M sodium citrate buffer (pH 2.2) to make a volume of 5.0 mL. The mixture was passed through a C-18 Sep Pak (Waters Co., Milford, MA, USA) cartridge and filtered through a 0.22 μm membrane filter (Millipore, Billerica, MA, USA). Amino acids were determined using an automatic amino acid analyzer (Biochrom-20, Pharmacia Biotech Co., Uppsala, Sweden).
10
HPLC Analysis of Sugars and Organic Acids
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Sugar and organic acid contents were determined by high-performance liquid chromatography (HPLC), according to the method of Toshima et al. [23 (link)]. Each fraction (0.02 g) was dissolved in 5 mL of ultrapure water and passed through a 0.22-μm membrane filter (Millipore, Bedford, MA, USA) prior to analysis. For sugar content analysis, the extracts were subjected to HPLC using a UF-Amino station system (Shimazu, Kyoto, Japan) equipped with a refractive index detector (RID-10A, Shimadzu) and Asahipak NH2P-50 4E column (Showadenko, Tokyo, Japan). The chromatographic conditions were as follows: solvent, 75% (v/v) acetonitrile (CH3CN); column temperature, 40 °C; flow rate, 1.0 mL/min. Retention times and spectra were compared with those of pure standards of glucose, fructose, and sucrose. For organic acid content determination, each fraction was analyzed by reverse-phase HPLC using a Prominence LC solution system (Shimadzu) equipped with a photodiode array detector (SPD-M20A, Shimadzu) and Inertsil ODS3 column (Shimadzu). The chromatographic conditions were as follows: solvent, 75% (v/v) CH3CN; column temperature 40 °C; flow rate, 1.2 mL/min. Retention times and spectra were compared with those of pure standards of quinic acid, citric acid, and malic acid. Results are expressed as milligrams per gram dry weight of each fraction. Sample extracts were analyzed three times.
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