Gel doc 2000 system

For 23 days, NHOst cells (3 × 10⁵) were cultured in T-25 flasks containing basal or supplemented medium and exposed to HA+AA solution. Then, cells were washed thrice with cold PBS and lysed with 2.5 mL of TRIzol™ reagent. Briefly, after 5 min of incubation, cell lysates were harvested and exposed to 0.5 mL of chloroform, and were then centrifuged at 12,000× g for 15 min at 4 °C. Then, the upper aqueous phase was transferred in a new tube and mixed with 1.25 mL of isopropyl alcohol. After another centrifugation step, the RNA pellet was resuspended in 75% ethanol. Spectrophotometric measurements were performed to assess the RNA concentration and to choose the proper volume for the reverse transcription of 2 μg of RNA [23 ]. In this respect, the SuperScript™ III First-Strand Synthesis System was exploited as recommended by the manufacturer. PCR experiments were performed with 1 µL of the synthetized cDNA as a template, following the thermal cycles reported in Table 1. The PCR products were loaded on 4% agarose gels and stained with ethidium bromide. Gel pictures were captured by the Gel Doc 2000 system (Bio-Rad Laboratories, Segrate, MI, Italy) and analyzed using the Quantity One Software v. 4.6.7 (Bio-Rad Laboratories, Segrate, MI, Italy).

Cell lysates of wild-type and mutant strains of V. vulnificus were prepared, and 60-μg quantities of protein extracts were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein pools in the supernatants of the same cultures used for collecting the cell lysates were precipitated by treatment with 10% (vol/vol) trichloroacetic acid, and 5-μg quantities of proteins in the supernatant concentrates were fractionated by SDS-PAGE. Blotted Hybond polyvinylidene difluoride (PVDF) membranes were incubated with polyclonal antibodies raised against various recombinant proteins, including flagellins, FHPs, IIA^Glc, or OmpU (1:5,000 [vol/vol]), followed by alkaline phosphatase-conjugated rabbit anti-rat IgG (1:5,000 [vol/vol]). Immunoreactive bands were visualized using the nitroblue tetrazolium (NBT)–5-bromo-4-chloro-3-indolylphosphate (BCIP) system (Promega). The intensities of the protein bands were quantified using a densitometer (Bio-Rad; Gel Doc 2000 system).

DNA supercoiling plays a key role in gene expression and genome organization [79 (link)]. The agarose gel electrophoresis assay was used to check whether the tested complexes 3a–3c are able to cleave DNA. Supercoiled plasmid pUC57 DNA (4634 bp); Thermo Scientific, ABO Sp. z o.o., Gdansk, Poland), was incubated with tested compounds (100, 200 and 300 µM). Next, loading buffer (1 μL) containing 25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol was added to allow the samples to be loaded into 1% w/v agarose gel. Electrophoresis was performed at 75 V in Tris-acetate- EDTA (TAE) buffer. The gel was stained using Midori green advance DNA stain (Genetics, ABO Sp. z o.o., Gdansk, Poland), and visualised under UV light with the BioRad Gel Doc 2000 system using LABWORK software (Hercules, California, USA). All experiments were carried out in triplicate under the same conditions.

The clonal relationships between O104 STEC strains isolated from cattle feces were assessed by PFGE typing. The human clinical strains, German (O104:H4; BAA-2326) and Montana outbreak (O104:H21; BAA-178) and O104:H7 strains (06–3637, 07–3598, 08–4061, 2011C-3665, 2012C-3400; provided by Nancy A. Strockbine, Centers for Disease Control and Prevention, Atlanta, GA) were also included for comparison. The PFGE was performed according to the Centers for Disease Control and Prevention’s PulseNet protocol. Briefly, agarose embedded DNA of the O104 isolates were digested with XbaI followed by separation of restriction fragments by electrophoresis. Salmonella enterica serotype Braenderup (strain H9812) DNA digested with XbaI was used as DNA marker. Gel images were captured with a Gel Doc 2000 system (Bio-Rad). PFGE patterns were analyzed using the Bionumerics software (Applied Maths, Inc., Austin, TX). Band-based Dice similarity coefficients and unweighted pair-group method for clustering with a position tolerance setting of 1.5% were used for optimization and band comparison. Isolates with 100% homology were grouped as subtypes and those isolates which were > 96% but less than 100% homologous were grouped as types based on the Dice coefficients.

Renal cortex was homogenized in lysis puffer containing 0.5% (v/v) NP40 (Nonidet P40), 137 mM NaCl, 2 mM EDTA, 50 mM Tris/HCl, pH 7, and 10% (v/v) glycerol. Following centrifugation, supernatants were stored at − 80 °C. For western blot analysis, 30 μg protein was fractionated by SDS/10% PAGE and blotted on to a nitrocellulose membrane. For detection of TNFα, the antibody was purchased from Genzyme (IP-400). A HRP-conjugated goat-anti-rabbit antibody and a POD-conjugated rabbit-anti-goat antibody (Dianova, Hamburg, Germany) were used as secondary antibodies. Western blot was developed using an ECL® system (Amersham Bioscience, Freiburg, Germany). Semiquantitative evaluation was performed using the Gel Doc 2000 System (Bio-Rad Laboratories, München, Germany).

S1-PFGE and Southern blot analysis were performed to estimate the size of the plasmid carrying the mphA gene. In brief, the isolates were embedded in 10 g/L Seakem Gold gel, digested with endonuclease S1 nuclease (TakaRa, Dalian, China) for 15 min at 37 °C. Chromosomal DNA of Salmonella serotype Braenderup (H9812) digested with XbaI was used as reference markers. Electrophoresis was run on a CHEF MAPPER variable angle system (Bio-Rad, California, America) with the parameters set at 0.22 s ~ 26.29 s at 6 V/cm for 15 h. Images were captured using a Gel Doc 2000 system (Bio-Rad) and converted to TIF format files for further analysis. Captured images were imported into the BioNumerics software version 6.0 database for processing and analysis. Cluster analysis used an unweighted pair group method with arithmetic mean (UPGMA). DNA fragments are transferred horizontally to nylon membrane (Millipore, USA) and hybridized with digoxin-labeled probes obtained by PCR amplification.