Terminal deoxynucleotidyl transferase

DNA strand breaks in retinal explant sections and 661 W cells were detected by terminal dUTP nick end-labeling (TUNEL) on fixed tissue and cells. Retinal sections or cells were permeabilized with 0.1% Triton X for 2 min followed by incubation with terminal deoxynucleotidyl transferase (Promega, Wisconsin, US) and fluorescein-12-dUTP (Roche, Risch-Rotkreuz, Switzerland) according to manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 (1:10,000) (Sigma). Sections and cells were incubated at 37 °C for 1 h in a humidified chamber and following several washes in 1xPBS, were mounted in Mowiol. Sections were viewed under a fluorescence microscope (Leica DM LB2). Eliminating the TdT enzyme served as a negative control (Supplementary Figure S1). In co-culture assays, TUNEL-positive 661 W cells could easily be distinguished from microglia based on nuclear size and phase contrast of cell preparations. Fluorescence intensity measurements of TUNEL in the ONL was performed using ImageJ software. An identical size box was drawn within the boundaries of the ONL for each explant and used to take five independent measurements, to be averaged.

Fourteen micron sagittal sections (lateral 2.90 mm, bregma 0.48 mm, interaural 9.48 mm) of frozen rat brain hemispheres were collected on glass slides (3 sections/slide) and subjected to pre-hybridization treatment using an established ISHH protocol as previously described^{44 (link)}. Oligodeoxyribonucleotide cDNA probes complementary to: GluN1 (bases 746–780, NM008169.1), GluN2A (bases 1642–1676, NM008170.2) or GluN2B (bases 1758–1792, NM010350.2) were 3′-end labelled with [³⁵S]-dATP using terminal deoxynucleotidyl transferase (Promega, UK). Probes were then diluted in hybridization buffer, pipetted onto the tissue sections (1 × 10⁶ cpm/section), cover-slipped and then incubated for over 16 h at 34 °C in lidded Perspex trays lined with filter paper soaked with 4× SSC/50% formamide. Post-hybridization washes included 2× SSC rinse at room temperature to remove cover-slips, 3× in 0.5× SSC for 20 min at 55 °C, and 2× in 0.5× SSC for 30 min at room temperature. Slides were rinsed in ddH₂O, dried and exposed to X-ray film (Kodak, Biomax MS) for 7–28 days with ¹⁴C-microscales. Average grey densities over the frontal cortex, and dentate gyrus, CA1 and CA3 subfields of the hippocampus in the three sections from each group were measured using computer-assisted image analysis. Densities were calibrated to ³⁵S nCi/g tissue equivalents using commercial ¹⁴C-microscales.

A total of four biological replicates of F0 and F1 each were used. Cells were grown to OD600 = 0.5 (mid-log) and 10 mL aliquots of culture were treated with 20 mL RNAProtect bacteria reagent (Qiagen) according to manufacturer’s protocol. RNA was isolated from these cells using RNAeasy MIDI kit (Qiagen). RNA was then converted to cDNA using Superscript II (Invitrogen). The cDNA was fragmented using DNAseI (New England Biolabs) and then labelled with Genechip DNA labeling reagent (Affymetrix) and terminal deoxynucleotidyl transferase (Promega). The hybridization cocktail was prepared using the GeneChip Hybridization, Wash and Stain Kit (Affymetrix) and hybridized to a P. aeruginosa genome array (Affymetrix). All steps performed were carried out according to the respective manufacturers’ protocol. Arrays were hybridized for 16 h at 50°C and then washed and stained with the GeneChip Hybridization, Wash and Stain Kit (Affymetrix). Data from the arrays were analyzed using Partek Genomics Suite. A list of differentially expressed genes (DEGs) was constructed using the criteria of p < 0.05 (one-way ANOVA) and a fold change (FC) of 1.5. Data are deposited in the Gene Expression Omnibus (GEO) database under accession number GSE75654.

For the identification of the dcw operon transcription start site in B. cenocepacia J2315, 5’ rapid amplification of cDNA ends (5’-RACE) was performed. The RNA extraction, DNase treatment and reverse transcription were carried out as mentioned above, using the primer mraZRACErev1 (Table S2) for the RT-PCR. Subsequently, to degrade the RNA of the resulting cDNA-RNA heteroduplex and improve the further step yield, the sample was treated with ribonuclease H (Promega, Madison, WI, USA) adding 2 U of enzyme directly in 20 uL of RT-PCR mix after the reverse transcription reaction and incubating at 37 °C for 30 min. Then, the cDNA was purified and a tail of polyadenosine was added to the 3’ end by terminal deoxynucleotidyl transferase (Promega) reaction, according to the manufacturer’s instructions. The cDNA was amplified by PCR using the polyT universal forward primer RA1 (Table S2) coupled with the reverse primer mraZRACErev2 (Table S2). The reaction product of the first PCR was used as template for the nested PCR performed with the primers RA2 and mraZRACErev3 (Table S2), designed to amplify a fragment within the first product sequence. The resulting amplified fragment was sequenced to identify the dcw cluster transcription start site.

DNA strand breaks in retinal cell nuclei and 661W cells were detected by terminal dUTP nick-end labeling (TUNEL) on fixed tissue and cells. Frozen retinal sections or coverslips with cells were permeabilzed with 0.1% Triton X for 2 min followed by incubation with terminal deoxynucleotidyl transferase (Promega, Wisconsin, US) and fluorescein-12-dUTP (Roche, Risch-Rotkreuz, Switzerland) according to manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 (1μg/mL) (Sigma). Sections and cells were incubated at 37°C for 1 h in a humidified chamber and following several washes in 1xPBS, were mounted in Mowiol. Sections were viewed under a fluorescence microscope (Leica DM LB2). In co-culture assays, TUNEL-positive 661W cells could easily be distinguished from microglia based on nuclear size and phase contrast of cell preparations. For each treatment on mice, at least three animals were used and three fields (x40 magnification) per section of at least three different sections were evaluated. For each treatment on coverslips, 6 coverslips were used per treatment and 50–200 661W cells analyzed per coverslip for TUNEL positivity.