Bacteriophage T4 (DSM-4505) and its E. coli host strain (DSM-613) were obtained from the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Braunschweig, Germany). Bacterial cultures were grown and maintained on Luria-Bertani (LB) broth. Bacteriophages were amplified in host bacteria according to [16 (link)]. Briefly, 10 mL of LB broth was inoculated with a single colony of E. coli and incubated overnight at 37 °C (150 rpm). Then, 5 mL of phage lysate was added and the phage-bacteria culture was incubated for the subsequent 24 h in the same conditions. The culture was centrifuged (5000 rpm, t = 10 min, 20 °C) and the supernatant was filtered through a 0.22 µm syringe filter (Merck Millipore, Darmstadt, Germany). The filtrate was introduced into a 20-h host culture in 100 mL of liquid LB medium and incubated for 24 h at 37 °C with shaking (150 rpm). The culture was centrifuged (5000 rpm, t = 10 min, 20 °C) and filtered through a 0.22 µm syringe filter (Merck Millipore, Germany). Phage titer was determined by the double-agar-layer method (plaque assay) [17 (link)].
0.22 m syringe filter
Manufactured by Merck Group
Sourced in United States, Germany, Poland, United Kingdom
The 0.22 µm syringe filter is a laboratory equipment used for filtration. It is designed to remove particles and microorganisms from liquid samples, with a pore size of 0.22 micrometers.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Dsm 613, by Leibniz Institute DSMZ (1 mentions)
Ethephon, by Merck Group (1 mentions)
Nexion 300x, by PerkinElmer (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Nano zsp, by Malvern Panalytical (1 mentions)
Icp aes, by Shimadzu (1 mentions)
Baicalein, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Genejuice, by Merck Group (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Potassium cyanide kcn, by Merck Group (1 mentions)
Matlab r2012b, by MathWorks (1 mentions)
Vibra cell vcx130, by Sonics (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Auranofin, by Merck Group (1 mentions)
Af2200, by Eppendorf (1 mentions)
Zetasizer nano zs machine, by Malvern Panalytical (1 mentions)
Exoquick tc isolation kit, by System Biosciences (1 mentions)
41 protocols using 0.22 m syringe filter
1
Bacteriophage T4 Amplification and Titration
2
Cadmium Stress Response in Plant Roots
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Freshly subcultured roots were incubated for 21 days to obtain at least 1.5 g of fresh weight. Afterwards, they were weighed and transferred to 100 mL of fresh medium five days prior to elicitation. A 50 mM stock solution of CaCl2 was prepared, then the solution was sterilized by filtration through a 0.22 m syringe filter (Millipore, Bionovo, Legnica, Poland) and added to the culture medium to obtain the final concentrations of 25 µmol/L. The cultures were cultivated with cadmium ions for 3, 7, 14, and 21 days.
3
Elicitation of Secondary Metabolites
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Freshly subcultured roots were incubated for 15–23 days to obtain at least 1.5 g of fresh weight. Afterwards, they were weighed and transferred to a fresh medium five days prior to elicitation. Appropriately weighed samples of ethephon (2-chloroethylphosphonic acid; Sigma-Aldrich, St. Louis, MO, USA) and ABA (DuchefaBiochemie, Netherlands) were used to prepare stock solutions of 0.1 mol/L in distilled water and 96% ethanol, respectively. Further, ABA solutions were prepared in distilled water. The solutions were sterilized by filtration through a 0.22 m syringe filter (Millipore, Bionovo, Legnica, Poland) and added to the culture medium to obtain the final concentrations of 10 and 100 µmol/L. The control cultures (in ABA treatment experiments) were treated with an adequate volume of 96% ethanol. The incubation period before harvesting the roots and the medium lasted five days.
4
Quantifying Gold Nanoparticle Uptake
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A549 cells were seeded in six well plates at a density of 2 × 105 cells/well in 3 mL of CCM, supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C. At 24 h post-seeding, the cells were pre-treated with either 10 µg/mL of chlorpromazine (clathrin-coated pit inhibitor), 5 µg/mL of nystatin (caveolae-mediated inhibitor), 2.5 µM of methyl-β-cyclodextrin (MβCD) (caveolae-mediated/cholesterol inhibitor), and 10 µg/mL of wortmannin (macropinocytosis inhibitor) for 1 h. This was followed by treatment with 30 µg/mL of different sizes of AuNPs dispersed in CCM for 3 h. After exposure, the cells were washed three times with PBS, harvested, and transferred to a glass vial. The cells were digested with 3 mL of aqua regia prepared with 37% HCL and 70% HNO3. Then, 1 mL of hydrogen peroxide was added to the cell suspension. The glass vials were properly closed and heated at 120 °C for 30 min. The samples were diluted to a final acid concentration of 2% HNO3 and filtered with a 0.22 µM syringe filter (Sigma Aldrich, UK) to ensure the removal of any debris. The total Au concentration in the samples was measured using the same procedure as described in Section 2.5.1 .
5
Quantifying Gold Nanoparticle Uptake in A549 Cells
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ICP-MS was used to measure the amount of Au uptake by A549 cells. A549 cells were seeded in six well plates at a density of 2 × 105 cells/well in 3 mL of CCM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C. At 24 h post-seeding, the A549 cells were exposed to 5, 15, and 30 µg/mL of different sizes of AuNPs dispersed in CCM for 24 h. After the exposure, the cells were washed three times with PBS, harvested, and transferred to a glass vial (Ampulla, Lancashire, UK). The cells were digested with 3 mL of aqua regia prepared with 37% hydrochloric acid (HCl) and 70% nitric acid (HNO3), then 1 mL of hydrogen peroxide was added to the cell suspension. The glass vials were properly closed and heated at 120 °C for 30 min. The samples were diluted to a final acid concentration of 2% HNO3 and filtered with a 0.22 µM syringe filter (Sigma Aldrich, UK) to ensure the removal of any debris. The total Au concentration in the samples was measured by ICP-MS (NexION 300X, Perkin Elmer, Waltham, MA, USA) in the Helium KED mode (Helium Gas Flow at 5 mL/min). A calibration curve of gold standard series was also prepared (0, 0.25, 0.5, 1, 2, 5, 10, 20, 50, and 100 ppb) in 2% HNO3 to set up the ICP-MS.
6
Inhibiting Quorum Sensing in P. aeruginosa
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P. aeruginosa PAO1 cultures were grown overnight in LB medium to yield an OD600 of 2.0 and then incubated with appropriate concentrations (32–128 µg/mL) of baicalein for 12 hours or in the absence of agent as a blank control. The P. aeruginosa PAO1 QS-mutant deficient in lasI-rhlI was grown along with the treated and untreated P. aeruginosa PAO1 cultures, and it served as a negative control. Cells were harvested by centrifugation and washed twice with sterile phosphate-buffered saline (PBS) for RNA extraction, and the supernatants were sterilized by filtering through a 0.22 µm syringe filter (EMD Millipore) before being either used immediately or stored at −80°C for later measurements of pyocyanin, LasA protease, LasB elastase, rhamnolipids, and AHLs. To prepare the inocula for the motility, adhesion, and biofilm assays, the overnight culture medium was adjusted to a cell density of 1×107 CFU/mL based on counting colonies on the LB agar plate.
7
Ragweed and Dust Mite Extract Preparation
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Aqueous ragweed pollen extracts (RWE) were prepared as previously described (46 (link)). Briefly, ragweed pollen were suspended in PBS (2.5 mg/ml), incubated for 30 min at 37°C and then centrifuged for 10 min at 4000 rpm by 4°C. The supernatants were sterile filtered through a 0.22 µm syringe filter (Merck KGaA, Darmstadt, Germany) and frozen at -80°C. House dust mite extracts from Dermatophagoides farinae (Stallergene Greer Laboratories, Kamp-Lintfort, Germany and Citeq BV, Groningen, NL) were suspended in 9% NaCl (1 mg/ml) and stored at -20°C until use. The final solution for intranasal instillation was prepared in PBS.
8
Synthesis and Characterization of AB-Lac Nanoparticles
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The mixture of PSar106-block-PLLA32 in chloroform (1 mL, 1 µmol) and 100 µL of B cluster (10 µmol) in chloroform (or DMF) were added to a test tube and evaporated to form a polymer-B compound film. Then, 2.0 mL of DPBS was added and treated with a bath-type sonicator for 20 min, followed by conditioning the mixture with the PD-10 desalting column (GE Healthcare, Buckinghamshire, UK) and the nanoparticle-enriched eluate was collected. The eluate was then passed through a 0.22 µm syringe filter (Merck Millipore, Dublin, Ireland), followed by filtration at 0.1 µm (PALL Corporation, NY, USA) to exclude larger-sized and aggregated particles (Figure 1 C). The B compounds loaded into the AB-Lac particles comprised o-carborane, m-carborane, p-carborane, diC1-Carb, diC6-Carb, or diC12-Carb. The loaded B amount in the particles was measured with ICP-AES (Shimadzu, Kyoto, Japan). The particle size distribution (PSD) and polydispersity index (PdI) of the AB-Lac particles were determined by taking 40 µL of sample solution into a disposable low volume cuvette (GmbH & Co. KG, Weinheim, Germany) and a Zetasizer (Nano ZSP; Malvern, Instruments, Malvern, UK) for 60 s equilibration time. The PSD and PdI data are represented as mean ± S.D.
9
Baicalein Evaluation of P. aeruginosa
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This study used the sequenced P. aeruginosa PAO1 wild-type strain and its mutants deficient in lasR-rhlR (ΔlasI-ΔrhlI), generously donated by Dr Liang Yang (Nanyang Technological University, Singapore). Strains were routinely stored in Luria–Bertani (LB) broth containing 25% glycerol at −80°C. baicalein standard dry powder purchased from Sigma-Aldrich (St Louis, MO, USA) with a purity of ≥98% by high-performance liquid chromatography (HPLC) was freshly dissolved in dimethyl sulfoxide (Sigma-Aldrich). The baicalein stock solutions were sterilized by passing through a 0.22 µm syringe filter (EMD Millipore, Billerica, MA, USA), and aliquots were stored at −80°C until use.
10
Lentiviral Transfection Optimization for Eosinophils
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To ensure the safety and the titer of the pLVX-ShRNA2-m vector and the constructed pLVX-mCCR3-1+2+3+4-shRNA, these two lentiviruses were separately co-transfected with the packaging plasmids, Baculo p35, pCMV R8.2 and VSV (quantities: 2 µg Baculo p35, 2 µg VSV plasmid, 4.7 µg pCMV R8.2 plasmid and 2.3 µg Lentiviral vector), into 293T cells in 100-mm tissue culture dishes of DMEM containing 10% FBS without antibiotics at 37°C. The medium was replaced 24 h later, and the virus-containing medium was harvested 48 h following transduction. The supernatants were filtered through a 0.22 µm syringe filter (EMD Millipore, Billerica, MA, USA). The eosinophil cultures on day 10 were infected with the supernatants at a multiplicity of infection of 50, and polybrene (Sigma-Aldrich; Thermo Fisher Scientific, Inc.) was added to a final concentration of 8 µg/ml. The harvested eosinophils were transfected with either a blank control (RPMI 1640 medium), empty vector (pLVX-shRNA2-m) or the constructed target plasmid (pLVX-mCCR3-1+2+3+4-shRNA), respectively. The culture medium was aspirated 48 h following transduction and the cells were washed with PBS for the subsequent quantitative (q)PCR and western blot analyses, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assays.
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