Iron-free enterobactin (Escherichia coli), deferoxamine mesylate (DFO), pyoverdine (Pseudomonas fluorescens), ferrichrome (Ustilago sphaerogena), ferric chloride, Histopaque®−1077 and 1119, RPMI, Triton X-100, PIPES, DMSO, mannitol, sucrose, EGTA, 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA), fatty acid-free bovine serum albumin (BSA) and Lipopolysaccharide (LPS) were purchased from Sigma (St Louis, MO). FITC Annexin-V apoptosis detection kit was purchased from BD Pharmingen (BD Biosciences, San Jose, CA) and Griess reagent kit from Molecular Probes (Life Technologies, Columbus, OH). SYBR® Green mix and qScript cDNA synthesis kit were procured from Quanta Biosciences (Beverly, MA). Duoset enzyme-linked immunosorbent assay (ELISA) kits for mouse lipocalin 2 (Lcn2), and interleukin (IL)-6 were obtained from R&D Systems (Minneapolis, MN). Chrome azurol S (CAS) was purchased from Acros Organics (Geel, Belgium).
Chrome azurol s
Manufactured by Thermo Fisher Scientific
Chrome azurol S (CAS) is a chemical compound used as a colorimetric reagent in various analytical techniques. It is commonly employed in the detection and quantification of metal ions, particularly iron, in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Ferrichrome, by Merck Group (3 mentions)
Pyoverdine, by Merck Group (3 mentions)
Pipes, by Merck Group (3 mentions)
Roswell park memorial institute (rpmi), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Histopaque 1077 and 1119, by Merck Group (2 mentions)
Ferric chloride, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
5 sulfosalicylic acid, by Merck Group (2 mentions)
Hdtma, by Oakwood Chemical (2 mentions)
Piperazine hexahydrate, by Merck Group (2 mentions)
Iron 3 chloride hexahydrate, by Merck Group (2 mentions)
Mannitol, by Merck Group (1 mentions)
Griess reagent kit, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Merck Group (1 mentions)
Sybr green mix, by Quanta Biosciences (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (1 mentions)
Fatty acid free bovine serum albumin, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
7 protocols using chrome azurol s
1
Siderophore-Mediated Iron Acquisition Assays
2
Enterobactin and Siderophore Assays
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Enterobactin (Ent; from Escherichia coli) procured from Sigma-Aldrich (St. Louis, MO) is supplied free of iron and endotoxin (≥98% purity; HPLC). Iron-free Pyoverdine (from Pseudomonas fluorescens), ferrichrome (from Ustilago sphaerogena), Deferiprone, deferoxamine mesylate salt (DFO; from Streptomyces pilosus), 2,3 dihydroxybenzoic acid (2,3-DHBA), lipopolysaccharide (LPS) from E. coli 0128:B12, dimethyl sulfoxide (DMSO), ferric chloride (FeCl3, Fe3+), 2,2ʹ-dipyridyl, and cyclosporin H were purchased from Sigma-Aldrich (St. Louis, MO). Flagellin (FliC) from Salmonella enterica subsp. enterica serovar Typhimurium (SL3201, fljB-), a kind gift from Dr. Andrew Gewirtz, Georgia State University, was purified through sequential cation and anion-exchange chromatography as previously described.52 (link) FITC Annexin-V apoptosis detection kit was purchased from Molecular Probes (Life Technologies, Columbus, OH). Chrome azurol S (CAS) was purchased from Acros Organics (Geel, Belgium). Recombinant mouse Lcn2 (alias neutrophil gelatinase-associated lipocalin) (rec-Lcn2; Cat# CM17) produced by a mammalian (human) expression system was procured from Novoprotein Scientific Inc. (Fremont, CA). Carrier-free rec-Lcn2 was supplied at a purity ≥95% as determined by reducing SDS-PAGE and free from endotoxin, siderophore, and iron.
3
Siderophore-Mediated Bacterial Interactions
4
Preparation of CAS-Fe3+ Solution
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The CAS-Fe3+ solution was prepared as described by Schwyn and Neilands with minor modifications.[35 (link),38 (link)] All glassware was rinsed with 6 M hydrochloric acid, followed by double distilled water prior to use. In a graduated glass cylinder 6 mL of a 10 mM solution of hexadecyltrimethylammonium bromide (HDTMA, Oakwood Chemical) was diluted to approx. 40 mL with double distilled water. While stirring, 1.5 mL of a ferric iron solution (1 mM iron(III) chloride hexahydrate (Sigma–Aldrich) in 10 mM hydrochloric acid) was added. Upon slow addition of 7.5 mL of 2 mM aqueous Chrome Azurol S (dark orange, ThermoFisher Scientific) the solution adopted an intense blue coloration. In a separate glass flask, 9.71 g piperazine-hexahydrate (Sigma–Aldrich) were dissolved in double distilled water (ca. 20 mL) and cooled on ice. 6.25 mL of 12 N hydrochloric acid were carefully added and this buffered solution was poured into the glass cylinder containing the CAS-Fe3+ solution before double distilled water was added to a final volume of 100 mL. Finally, 5-sulfosalicylic acid (Sigma–Aldrich) was added to a final concentration of 4 mM and the solution was stored in a light-protected polyethylene bottle at room temperature. This CAS-Fe3+ solution was used for all in vitro experiments over the course of several weeks.
5
Preparation of CAS-Fe3+ Solution
6
Silica-Titania Xerogel Synthesis and Characterization
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The following reagents were purchased from Acros Organics: triphenylmethane dyes (TPMD): pyrocatechol violet (PV), eriochrome cyanine R (ECR), chrome azurol S (CAS), hydrochloric acid, titanium(IV) tetraethoxyde, tetraethyl orthosilicate, and oxalic acid. All the reagents were of analytical grade, titanium(IV) tetraethoxyde was of technical grade. Stock solutions of oxalic acid and TPMD were prepared with doubly distilled water. Only freshly prepared solutions were used.
Silica–titania xerogels were obtained by drying in Ethos microwave equipment (Milestone, Sorisole, Italy). Surface area, porosity BET analysis, and BJH pore distribution analysis were carried out with ASAP 2000 (Micromeritics, Norcross, GA, USA). Absorbance of solutions (l = 1.0 cm) and xerogels water suspensions (l = 0.1 cm) was measured using Lambda 35 spectrophotometer (PerkinElmer, Waltham, MA, USA) equipped with 50 mm integrating sphere (Labsphere, North Sutton, NH, USA). The pH value was measured using Expert-001 (Econix Expert, Moscow, Russia) potentiometer with pH sensitive electrode. Chromatographic determination was conducted using MAESTRO HPLC system (Interlab, Moscow, Russia). A Kromasil C18 column (250 × 4.6 mm, 5 μm particle size) served as the stationary phase, mobile phase was distilled water (pH 2). UV detection was performed at 220 nm and a flow rate of 1.0 mL·min−1.
Silica–titania xerogels were obtained by drying in Ethos microwave equipment (Milestone, Sorisole, Italy). Surface area, porosity BET analysis, and BJH pore distribution analysis were carried out with ASAP 2000 (Micromeritics, Norcross, GA, USA). Absorbance of solutions (l = 1.0 cm) and xerogels water suspensions (l = 0.1 cm) was measured using Lambda 35 spectrophotometer (PerkinElmer, Waltham, MA, USA) equipped with 50 mm integrating sphere (Labsphere, North Sutton, NH, USA). The pH value was measured using Expert-001 (Econix Expert, Moscow, Russia) potentiometer with pH sensitive electrode. Chromatographic determination was conducted using MAESTRO HPLC system (Interlab, Moscow, Russia). A Kromasil C18 column (250 × 4.6 mm, 5 μm particle size) served as the stationary phase, mobile phase was distilled water (pH 2). UV detection was performed at 220 nm and a flow rate of 1.0 mL·min−1.
7
Recombinant Protein Procurement for Biochemical Assays
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Mouse recombinant (rec)-Lcn2 was obtained from Cell Signalling and human rec-NGAL was acquired from R&D Systems; both of which are free from endotoxin, siderophore, and iron. Human MPO was procured from R&D Systems (Minneapolis, MN). EGCG, PMA, ferric chloride, PIPES, agar, and H2O2 were procured from Sigma-Aldrich (St. Louis, MO). Bovine milk LPO was purchased from Worthington Biochemical Corp (Lakewood, NJ). Chrome Azurol S was purchased from Acros Organics. Guaiacol (2-methoxyphenol) was obtained from Alfa Aesar (Ward Hill, MA).
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