Sodium dodecyl sulfate (sds)

Proliferation was assessed using Thiazolyl Blue Tetrazolium Bromide (MTT) Assay. 2500 HN cells overexpressing MERTK or GFP were plated in 100 μl medium in 96-Well plates (Corning, Corning, NY, USA). 500 μg/ml MTT (Sigma-Aldrich, Munich, Germany) dissolved in phosphate buffered saline (PBS) (Gibco® Life technologies, Darmstadt, Germany) was added at different time points. Four hours after treatment with MTT 100 μl solubilization buffer (40 % vol/vol Dimethylformamide (Alfa Aesar, Karlsruhe, Germany), 2 % vol/vol glacial acetic (Merck, Darmstadt, Germany), 16 % wt/vol sodium dodecyl sulfate (Applichem, Darmstadt, Germany), pH 4.7) was added and absorbance at 595 nm was measured the next day. For inhibition experiments 5000 Detroit 562 or 2500 HN cells were plated in 50 μl medium before adding different amounts of UNC1062 (AOBIOUS, Gloucester, MA, USA) in 50 μl medium the next day. Knockdown cells were induced for 72 hours with 1 μg/ml doxycycline before plating 5000 cells in 100 μl medium. MTT was added after different time points as described above.
Each experiment was performed in triplicates and repeated at least three times.

The first step of developing rapid test protocols for the analysis of blood cultures was the evaluation of a suitable hemolytic agent for each test method. Therefore, the following hemolytic agents were assessed with 10 test isolates: saponin (Sigma-Aldrich, Steinheim, Germany), sodium dodecyl sulfate (SDS; AppliChem, Darmstadt, Germany), ammonium-chloride potassium lysis buffer (ACK; consisting of 150 mM KHCO₃ [Carl Roth, Karlsruhe, Germany] abd 10 mM NH₄Cl [Sigma-Aldrich]), and Triton X (Sigma-Aldrich). The impacts of the hemolysis protocols on results and readability were analyzed. Additionally, different sample volumes were compared to achieve the overall best sensitivity without giving rise to false-positive results. For the β-CARBA test, 10% SDS provided complete hemolysis without interference with the test. For the Carba NP test and NeoRapid CARB, 5% saponin showed the best results for hemolysis with minimal interference and was therefore selected for the study. Triton-X and ACK lysis buffer resulted in less complete hemolysis and/or gave rise to false-positive results (data not shown). After blood culture fluid was drawn for the different protocols and erythrocytes were lysed, the bacterial pellet was washed with water and phosphate-buffered saline (PBS; pH 7.4) for all protocols to optimize readability and pH values.

Folin-Ciocalteu reagent, sodium carbonate anhydrous (Na₂CO₃), gallic acid, sodium nitrite solution (NaNO), sodiumhydroxyde (NaOH), aluminum chloride hexahydrate solution (AlCl₃, 6H₂O), iron (III) chloride anhydrous (FeCl₃), and catechin were purchased from Fluka (Buchs, Switzerland). 2,2-diphenyl-1-picrylhydrazyl-(DPPH), 2,6-di-tert-butyl-4-hydroxytoluene (BHT), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonate (ABTS) were purchased from Sigma-Aldrich (Murcia, spain), Tris (hydroxymethyl)-aminomethane (TRIS), and l-cysteine of analytical grade were obtained from Merck, Darmstadt, Germany. 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) all of analytical grade were obtained from Fluka, Stenheim, Germany. Sodium dodecyl sulfate (SDS) was obtained from AppliChem GmbH, Darmstadt, Germany. Commercial mix was purchased from a supermarket (Murcia, spain).

Boric acid (≥ 99.5%), benzoic acid (≥ 99.0%), and dimethyl sulfoxide (99.9%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Ultra-pure water was of Millipore® grade (specific resistivity 18.2 MΩcm (25 °C)). Sodium hydroxide pellets (99.5–100.5%), LiChrosolv water for chromatography (≥ 99.0%), and formic acid (98.0%) were obtained from Merck (Darmstadt, Germany), acetonitrile (99.9%) from VWR (Radnor, PA, USA), and sodium dodecyl sulfate (≥ 99.5%) from AppliChem (Darmstadt, Germany). Birch pollen grains (Betula pendula) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), quercetin-3-O-sophoroside from Carbosynth (Newbury, UK), and Betv1a from Biomay (Vienna, Austria).

Feces samples were collected and stored at −20 °C until processing for DNA-based 16S rRNA gene sequencing. DNA was extracted using an established phenol–chloroform-based method^{65 (link)}. In short, 500 µl of extraction buffer (200 mM Tris (Roth), 20 mM EDTA (Roth), 200 mM NaCl (Roth), pH 8.0), 200 µl of 20% SDS (AppliChem), 500 µl of phenol:chloroform:isoamyl alcohol (PCI) (24:24:1) (Roth) and 100 µl of zirconia/silica beads (0.1 mm diameter) (Roth) were added to each feces sample. Samples were lysed and homogenized twice using a Mini-BeadBeater-96 (BioSpec) for 2 min. After centrifugation and additional extraction with PCI (24:24:1), DNA was precipitated using 500 µl isopropanol (J.T.Baker) and 0.1 volume of 3 M sodium acetate (AppliChem). Samples were incubated at −20 °C overnight and centrifuged at 4 °C at maximum speed for 20 min. The resulting DNA pellet was dried, resuspended in TE buffer (AppliChem) with 100 µg/ml RNase I (Sigma-Aldrich) and column purified (BioBasic Inc.) to remove PCR inhibitors.

Feces samples were collected and stored at– 20°C until processing for DNA based 16S rRNA gene sequencing. DNA was extracted using a phenol-chloroform-based method previously described [42 (link)]. In brief, 500 μl of extraction buffer (200 mM Tris (Roth), 20 mM EDTA (Roth), 200 mM NaCl (Roth), pH 8.0), 200 μl of 20% SDS (AppliChem), 500 μl of phenol:chloroform:isoamyl alcohol (PCI) (24:24:1) (Roth) and 100 μl of zirconia/silica beads (0.1 mm diameter) (Roth) were added per feces sample. Lysis of bacteria was performed by mechanical disruption using a Mini-BeadBeater-96 (BioSpec) for two times 2 min. After centrifugation, aqueous phase was passed for another phenol:chloroform:isoamyl alcohol extraction before precipitation of DNA using 500 μl isopropanol (J.T. Baker) and 0.1 volume of 3 M sodium acetate (Applichem). Samples were incubated at—20°C for at least several hours or overnight and centrifuged at 4°C at maximum speed for 20 min. Resulting DNA pellet was washed, dried using a speed vacuum and resuspended in TE Buffer (Applichem) with 100 μg/ml RNase I (Applichem). Crude DNA was column purified (BioBasic Inc.) to remove PCR inhibitors.

BEAS-2B cells were grown to confluence in 150 cm² and A549 cells were grown to at least 50% confluence in 75-cm² culture flasks (TPP, Trasadingen, Switzerland) prior to a 24 h treatment with the individual PAHs (B[a]P, Flthn, and 1-MeA) or B[a]P in combination with LMW PAHs. Test substances were first dissolved in DMSO and then further diluted in culture medium containing all supplements. The DMSO concentration in culture medium never exceeded 0.3% (v/v). These two cell types were chosen for these studies to represent upper airway and alveolar regions of the lung. After 24 h incubation, all test chemicals were removed, and cells were washed twice with PBS prior to detachment with trypsin-EDTA. Harvested cells were washed twice with PBS and centrifuged (250× g, 5 min, rt). After the last centrifugation step, the supernatant was discarded. Depending on the further use, cell pellets were either stored directly at −80 °C for adduct analyses or, for immunoblotting, were suspended in 300 µL RIPA buffer (0.1% v/v SDS (AppliChem, Darmstadt, Germany), 150 mM NaCl, 50 mM Tris-HCl, 1% v/v Nonidet™ P 40 Substitute (Sigma) and 0.5% w/v sodium deoxycholate (AppliChem) plus 1% v/v protease inhibitor cocktail (Sigma)). At least three independent experiments were conducted.