Ds 11 spectrophotometer fluorometer

For mRNA expression analysis, in vitro infection was performed as described above and HT-29/B6 samples were taken at 1, 5 and 24 h p.i. considering respective negative controls (non-infected cells). For this, cell monolayers were washed three times with PBS and lysed directly in each well of 8-well plates by addition of cell lysis buffer (Roboklon, Berlin, Germany). Total RNA was isolated with Universal RNA/miRNA Purification Kit (Roboklon) according to the manufacturer’s protocol. The RNA quality and quantity was controlled using the Agilent 2100 Bioanalyzer with the RNA Nano Chips (Agilent, Waldbronn, Germany) and the DS-11 Spectrophotometer/Fluorometer (DeNovix Inc., Wilmington, USA), respectively. Relative expression of mRNA coding for IL-6 and IL-8 was quantified by RT-qPCR as described earlier [54 (link)]. For normalisation of gene expression, ACTB and B2M transcripts were used as reference genes and relative gene expression was calculated as described earlier [54 (link)] by the ∆∆Ct method [55 (link)]. Experiments were performed in triplicate. Primers used in this study are listed in Table 1.

Archived isolates were revived and maintained on blood agar at 35°C under anaerobic conditions as described above. Growth was resuspended in 1.0 mL TSB and cells were pelleted by centrifugation at >5,000 × g for 10 min. Following removal of supernatant, genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions for Gram-negative bacteria. Extracted DNA was quantitated using the QuantiFluor dsDNA System (Promega) and a DS-11 Spectrophotometer/Fluorometer (DeNovix). Sequencing libraries were prepared using a DNA Prep Kit (Illumina) according to the manufacturer’s instructions and quantitated with the QuantiFluor dsDNA System. Paired-end sequencing was performed with a 500-cycle MiSeq reagent kit v2 on a MiSeq instrument (Illumina).

The mucus of the skin was swabbed from fish sacrificed at 0.6 °C following a similar procedure as described in Section 2.6.3, and the swabs were stored in sterile Falcon tubes at −80 °C until further processing. DNA was isolated from the swabs using the PureLink™ Microbiome DNA Purification Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the instructions of the manufacturer. DNA quality and concentration were controlled in a Denovix DS-11 spectrophotometer/fluorometer. Samples showing suitable DNA concentrations and quality ratios were submitted to high-throughput sequencing of the V4-V5 hypervariable region of the 16S rRNA gene on the Illumina MiSeq platform, as described in [30 (link)]. Briefly, 16S rRNA amplicons were amplified by PCR using the bacterial primer pair 515F/907R, further purified by a 1.2% agarose gel electrophoresis, and mixed at equimolar concentrations for sequencing. Sequence library preparation was done with the TruSeq Nano DNA LT Sample Prep Kit Set A, and the sequencing was performed with the MiSeq Reagent Kit v3 (600 cycles). All sequence data used in this study were placed in the European Nucleotide Archive with the accession number PRJEB56124.