P. aeruginosa WT and ΔbfmS, ΔbfmR, and ΔbfmRS mutants were grown in LB overnight at 37 °C, then 1 mL culture of each sample was centrifuged at 12,000 rpm for 2 min and resuspended in the iron-deficient succinate medium and grown for 48 h49 (link). For siderophore measurement, the supernatants were filtered through 0.45 μm PVDF syringe filter (Millipore), and the absorbance at 400 nm was measured. Meanwhile, the OD600 of cultures was also measured, and the final siderophore production was calculated as A400/OD600. As for P. putida KT2440 and Pseudomonas sp. MRSN12121, the bacteria were grown on a similar medium, and the supernatants were filtered through 0.45 μm PVDF syringe filter (Millipore) and used for Chrome Azurol S (CAS) assay50 (link). 1 mL supernatant of bacterial cultures was mixed with equivalent CAS buffer and cultured at 16 °C for 1 h, a color change from blue to orange was read at the absorbance 630 nm using UV Spectrophotometer, with blank culture medium as control. The standard curve was established with the standard PVDI samples (Sigma-Aldrich).
Pvdf syringe filter
Manufactured by Merck Group
Sourced in United States, Germany
The PVDF syringe filter is a type of lab equipment designed for the filtration of small liquid samples. It is made of polyvinylidene fluoride (PVDF), a durable and chemically resistant material. The primary function of this filter is to remove particulates and impurities from liquids prior to analysis or further processing, helping to ensure sample purity and integrity.
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Lab products found in correlation
Polybrene, by Merck Group (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Puromycin, by InvivoGen (2 mentions)
Platinum e plat e, by Cell Biolabs (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by BioLegend (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Casamino acids, by Avantor (1 mentions)
Na2hpo4, by Thermo Fisher Scientific (1 mentions)
Kh2po4, by Merck Group (1 mentions)
Agilent 1260 infinity, by Agilent Technologies (1 mentions)
Hplc grade, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Lc net 2 adc, by Jasco (1 mentions)
Dnazol, by Thermo Fisher Scientific (1 mentions)
Bioprime array cgh genomic labeling system, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Sodium phosphate dibasic dihydrate, by Merck Group (1 mentions)
Aztreonam, by Merck Group (1 mentions)
39 protocols using pvdf syringe filter
1
Siderophore Production Assay in Pseudomonas
2
Staphylococcus aureus Culture Supernatants
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S. aureus culture supernatants (CS) were generated in (i) CCY: 3% yeast extract (Fisher BioReagents) supplemented with 2% bacto-casamino acids (Amresco), 2.3% sodium pyruvate (Fisher BioReagents), 0.63% Na2HPO4 (Fisher BioReagents) and 0.041% KH2PO4 (Sigma), (ii) BHI (Fluka) and (iii) RPMI-CAS (RPMI-1640 medium (Gibco) supplemented with 1% casamino acids (Amresco). Overnight cultures obtained from a single colony were diluted to OD600nm = 0.03 in fresh culture medium and grown to early stationary phase for 8 hours. All tested S. aureus strains reached similar bacterial densities in the respective media. CS were generated by centrifugation (5000 × g, 4 °C), followed by filter sterilization (0.1 µm pore size PVDF syringe filters; Millipore).
3
HPLC Analysis of Phenolic Compounds
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The evaluations of substrate consumption and product formation in the CR and ER were performed by reversed phase high performance liquid chromatography (RP-HPLC) [16 (link)]. The analysis was carried out with an HPLC Agilent 1260 Infinity (Santa Clara, CA, USA) with a quaternary pump and diode array detector, and a C18 Zorbax SB-C18 5-µm column, measuring 4.6 cm × 150 mm. Data were processed with OpenLAB CDS software. The mobile phases employed were 2.5% acetic acid (A) and acetonitrile (B) (HPLC grade from Sigma-Aldrich) with an elution gradient from 0 to 10% B for 5 min, from 10 to 30% B for 20 min, and from 30 to 50% B for 20 min, flow 1 mL/min. In total, 20 µL of sample were injected, previously filtered through poly(vinylidene fluoride) (PVDF) syringe filters of 0.45 µm (Millipore, Burlington, MA, USA). Determinations were performed 10 min after the first aliquot of H2O2, then after every hour for 8 h and finally at 24 h. The detection of monomers and oligomers of catechin, epicatechin and resveratrol was performed at 250 and 280 nm.
4
Therapeutic Protein Purification Protocol
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Lysozyme was purchased from Fluka (Buchs, Germany), and a humanized
monoclonal antibody, isotype IgG1 [12 (link)], was used to model a therapeutic protein. Sucrose was
purchased from Sigma (Taufkirchen, Germany), Merck (Darmstadt, Germany), Caelo
(Hilden, Germany), VWR (Bruchsal, Germany) and donated by Südzucker (Mannheim,
Germany). PVDF syringe filters with a pore size of 0.2 and 0.1 μm were obtained
from Millipore (Schwalback, Germany), Anotop syringe filters with a pore size of
0.02 μm were obtained from GE Life Science (Freiburg, Germany).
monoclonal antibody, isotype IgG1 [12 (link)], was used to model a therapeutic protein. Sucrose was
purchased from Sigma (Taufkirchen, Germany), Merck (Darmstadt, Germany), Caelo
(Hilden, Germany), VWR (Bruchsal, Germany) and donated by Südzucker (Mannheim,
Germany). PVDF syringe filters with a pore size of 0.2 and 0.1 μm were obtained
from Millipore (Schwalback, Germany), Anotop syringe filters with a pore size of
0.02 μm were obtained from GE Life Science (Freiburg, Germany).
5
Quantification of Extracellular Metabolites
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Samples for quantification of extracellular metabolites were obtained by rapidly taking 2 ml of broth, followed by immediate removal of the cells by filtration through PVDF syringe filters with a pore size of 0.22 μm (Millipore, USA). When OD600 > 1, the culture broth was first centrifuged (3,600 g, 8 min) and the supernatant was similarly filtered before being stored in HPLC vials at −20°C for further analysis. Subsequently, samples were analysed by HPLC (Jasco, Japan) model LC‐NetII/ADC equipped with UV‐2075 Plus and RI‐2031 Plus detector. An Aminex HPX‐87H column (Bio‐Rad, kept at 65°C) was used and a solution of 0.01 M of H2SO4, with a flow rate of 0.5 ml/min, was used as the mobile phase. Quantitative analysis of desired compounds was performed by comparison with a mixture of standards with known concentrations of each metabolite. Calibration curves were prepared using the peak areas of the RI detector for glucose, glycerol, acetate, ethanol and succinate, and of the UV absorbance for malate and fumarate.
6
Genomic DNA Extraction and Labeling for ToxoGeneChip
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For each of the 6 strains tested, parasites were grown in human foreskin fibroblasts (HFFs), released from host cells by needle passage, washed once by centrifugation at 800 × g, and filtered through 5.0-µm polyvinylidene difluoride (PVDF) syringe filters (Millipore). Genomic DNA (gDNA) was harvested from purified parasites using DNAzol (Invitrogen) according to the manufacturer’s protocol and treated with RNase A to remove RNA contamination. After confirmation of purity by gel electrophoresis, gDNA was sheared as follows: 1 µg of gDNA was added to 750 µl of shearing buffer (Tris-EDTA [TE], pH 8.0, and 10% glycerol) and 1 µl of 20-µg/µl glycogen in a prechilled nebulizer (Invitrogen) on ice. Nebulization was performed at 40 lb/in2 for 3 min using pressurized nitrogen. The size range of the resultant gDNA fragments was confirmed to be 200 to 600 bp by gel electrophoresis. DNA fragments were precipitated using 100% isopropanol. Biotin labeling of DNA fragments was performed using the BioPrime Array CGH genomic labeling system (Invitrogen) according to the manufacturer’s protocol. The 10× dCTP nucleotide mix was used with biotin-dCTP. Purification of labeled fragments was performed with the BioPrime purification module. For each strain, 2 µg of labeled DNA was hybridized to the Affymetrix ToxoGeneChip.
7
Oligomeric State and Size Analysis
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The oligomeric state was analyzed by molecular sieving in a calibrated Superdex S‐75 column equilibrated with buffer A (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1 mM DTT). Samples of 1 mL of protein (0.2 mg/mL) in buffer A alone or with added ligand were loaded in the analytical column pre‐equilibrated in buffer A added with the corresponding ligand (0.3 mM). Additionally, we measured the hydrodynamic diameter by DLS of the proteins alone and complexed with their ligands at 30°C in a Zetasizer μV. Samples of protein (0.2 mg/mL) in buffer B (Tris–HCl 100 mM, pH 8.4, 300 mM NaCl, 1 mM DTT) were microfiltered through 0.22 μm polyvinylidene difluoride (PVDF) syringe filters (Millipore) before analysis. DLS values for each sample were averaged over 10 runs of 10 measurements per run (Figure S3B ).
8
Optimization of NISTmAb Capillary Electrophoresis
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Method development, optimization, and qualification were carried out on Primary Sample 8670 (PS 8670) derived from a single production lot of the NISTmAb. Bare fused silica capillaries (50 μm inner diameter, 67 cm total length) were purchased from Sciex Separations (PN 338451) and cut to 30.5 cm prior to use. SDS-MW gel buffer, acidic and basic wash solutions, MW ladder, 10 kDa internal standard, and Tris/SDS sample buffer (pH 9.0) were purchased as a kit from Sciex Separations (PN 390953). Citric acid (PN 27487-50G-F), sodium phosphate dibasic dihydrate (PN 71633-250G), pre-weighed iodoacetamide vials (PN A3221-10VL), 2-mercaptoethanol (PN M3148-25ML), and 20% (w/v) SDS solution (PN 05030–50 ± 0ML-F) were from Sigma Aldrich. L-histidine monohydrochloride (PN 2081–06) and L-histidine (PN 2080–05) were from J. T. Baker. Zeba columns used for buffer exchange were from Life Technologies (PN 89882). PVDF syringe filters were from EMD Millipore (PN SLSV025LS).
9
Isolation of Extracellular Vesicles via Ultrafiltration
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EVs were isolated from the media of cells following a 72 h incubation using a modified ultrafiltration procedure as previously described46 (link). Briefly, 100 mL of spent media per collection was sequentially passed through 0.22 µm PES and 0.1 µm PVDF syringe filters (Merck Millipore, Kilsyth, VIC, Australia). Clarified media was then placed in a 100 kDa centrifugal filter unit (Merck Millipore) and centrifuged at 4,000 × g, 4 °C until sample had passed through. The flowthrough was discarded and the retentate washed with 0.1 µm filtered sterile PBS twice to yield a retentate of concentrated EVs in PBS. Retentates were collected and stored at −30 °C until required.
10
Nanoparticle Filtration and Characterization
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