A Chirascan Circular Dichroism spectrophotometer (Applied Photophysics, Applied Photophysics Limited, Surrey, United Kingdom) was used to investigate the interaction between pyocyanin or phenazine and DNA. Mixtures of calf thymus double-stranded (ds) DNA (Sigma Aldrich, Australia) at 150 ng/μL with varying pyocyanin or phenazine (0, 25, 50 and 100 μM) concentrations in Milli-Q water were incubated at room temperature (25 ± 1°C) for 15 min. Following incubation, the absorbance of the mixture was scanned from 200–320 nm (at a scan rate of 0.5 s/nm) in a 1 mm path-length quartz cuvette. Shifts in the DNA absorbance peaks due to pyocyanin/phenazine binding was recorded and plotted as CD-mdeg vs. wavelength-nm.
Double stranded calf thymus dna
Manufactured by Merck Group
Sourced in Germany
Double-stranded calf thymus DNA is a laboratory reagent commonly used as a standard in various molecular biology applications. It consists of DNA extracted from the thymus gland of calves, with the DNA strands maintained in a double-helix structure. This product serves as a reference material for techniques such as DNA quantification, electrophoresis, and as a control sample in experiments involving nucleic acid analysis.
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Lab products found in correlation
Hoechst dye, by Merck Group (4 mentions)
Eucl3 6h2o, by Merck Group (3 mentions)
Gdcl3 6h2o, by Merck Group (3 mentions)
Tbcl3 6h2o, by Merck Group (3 mentions)
Smcl3 6h2o, by Merck Group (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Hoechst dye, by Thermo Fisher Scientific (3 mentions)
Fluoroskan ascent fl plate reader, by Thermo Fisher Scientific (2 mentions)
Human serum albumin (hsa), by Merck Group (2 mentions)
Lacl3, by Merck Group (2 mentions)
Chirascan circular dichroism spectrophotometer, by Applied Photophysics (1 mentions)
Golgiplug, by BD (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Nbt bcip, by Thermo Fisher Scientific (1 mentions)
Multiscreen elispot plates, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
16 protocols using double stranded calf thymus dna
1
Pyocyanin and Phenazine Binding to DNA
2
Inflammatory cytokine profiling in TMPD-induced mouse model
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WT mouse (host) was i.p. injected with TMPD and subsequently received 1–2x105 sorted Ly6ChighLy6G−CD11b+ cells from CD45.1 mice (donor) 2 weeks later. Six days later, spleen was harvested and cultured at 1 × 107 cells/mL in a 24-well bottom plate at 37 °C with 5 μg/ml of Golgiplug (BD Pharmingen) in the presence or absence of calf thymus double-stranded (ds) DNA (Sigma) at 100 μg/ml. Six hours later, cells were harvested and stained for CD45.1 and CD11c. Cells were fixed and permeabilized by incubation with cytofix/cytoperm solution (BD Pharmingen). Cells were then incubated with PE-anti-IL-6 (clone MP6-20F3), PE-TNF-α (MP6-XT22) (BD Pharmingen), PE-IL-1α (ALF-167), and PE-IL-1β (NJTEN3) (eBioscience) and analyzed on a FACSCalibur flow cytometer. Similar experiments were performed with various combination of hosts and donors.
In some experiments, WT and Irf7−/− mice were i.p. injected with TMPD. Two weeks later, peritoneal cells were harvested, cultured as above and stained with Alexa Fluor 488-Ly6C, PerCP-Cy5.5-Ly6G, and APC-CD11b. Cells were then fixed, permeabilized, and stained with anti-IL-6, TNF-α, PE-IL-1α, and PE-IL-1β Abs.
In some experiments, WT and Irf7−/− mice were i.p. injected with TMPD. Two weeks later, peritoneal cells were harvested, cultured as above and stained with Alexa Fluor 488-Ly6C, PerCP-Cy5.5-Ly6G, and APC-CD11b. Cells were then fixed, permeabilized, and stained with anti-IL-6, TNF-α, PE-IL-1α, and PE-IL-1β Abs.
3
ELISPOT Assay for Anti-dsDNA Plasma Cells
4
Polyreactivity Screening of Antibodies
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Antibodies were tested for polyreactivity by ELISA as previously described (Tiller et al., 2008 (link); Wardemann et al., 2003 (link)). High-protein binding plates were coated overnight at room temperature with six distinct antigens: 10 µg/ml calf thymus double-stranded DNA (Sigma; #D8515), 10 µg/ml single-stranded DNA prepared from double-stranded DNA (heated at 95°C for 30 min, immediately aliquoted, and frozen at −20°C), 10 µg/ml LPS from Escherichia coli (Sigma; #L2637), 10 µg/ml KLH (Sigma; #H8283), 10 µg/ml Cardiolipin (Sigma; #C0563), and 5 µg/ml human insulin (Sigma; I9278). Plates were washed, then blocked with PBS supplemented with 0.05% Tween and 1 mM EDTA before addition of mAbs diluted in DMEM (GIBCO BRL; #31331093) supplemented with 1% Nutridoma-SP (Roche; #11011375001). Plates were developed with HRP-labeled goat anti-human IgG Fc antibody (1:5,000; Dianova; #109-035-098) and 1-Step Ultra TMB ELISA Substrate Solution (Thermo Fisher Scientific; #34029) and stopped with 2 M sulfuric acid. In all assays, strongly polyreactive ED38 (Meffre et al., 2004 (link)) and weakly polyreactive eiJB40 (Wardemann et al., 2003 (link)) were used as positive controls and mGO53 as a negative control. The threshold for positive binding was calculated for each antigen separately as the OD450 subtracted by two SDs of eiJB40.
5
Determination of Antibody Polyreactivity
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Reactivity determination was done using high binding plates (Costar) coated with the following antigens at 3 μg/mL: calf thymus double-stranded DNA (Sigma); Histone (H1; Sigma); Ro52 (SSA-TRIM21; Sigma); LPS from Escherichia coli O55:B5 (Sigma); human Insulin (Lilly) and Ovalbumin (InvivoGen). Supernatants of highly confluent hybridoma cultures were diluted 1/2; HRP-conjugated goat anti-mouse IgG (Sigma) polyclonal antibody and Biotin rat anti-mouse IgM (R6-60.2, BD Biosciences) were used as detection antibodies and HRP-conjugated streptavidin (Roche) for the biotinylated antibody detection. Finally, microplates were developed with TMB (BD Bioscience) and reaction was stopped with 2M H2SO4. Microplates were read with an EPOCH Microplate Spectrophotometer (BioTek) at 450-570 nm. Antibodies which were positive for three or more antigens of our panel were considered polyreactive.
6
Characterization of NP Composition
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The NP samples were weighed (wet weight) and stored at −30°C until further use. After overnight lyophilization (Freezone 2.5, Labconco, Kansas City, Missouri, United States), the sample weights were measured (dry weight). The water content was calculated as (wet weight − dry weight)/wet weight. The lyophilized samples were digested overnight at 60°C in a papain solution (100 mM phosphate buffer, 5 mM L-cysteine, 5 mM ethylenediaminetetraacetic acid, and 140 μg/mL papain, all from Sigma). The DNA, sulfated glycosaminoglycans, and hydroxyproline contents were measured in each sample. The amount of DNA per sample was measured with a Hoechst 33528 assay as described by Cesarone et al,16 (link) using a standard curve of double-stranded calf thymus DNA (Sigma). The amount of sulfated glycosaminoglycans per sample relates to the proteoglycan content and was measured with a 1,9-dimethylmethylene blue assay as described by Farndale et al,17 (link) based on a standard curve of chondroitin-sulfate from shark cartilage (Sigma). The amount of hydroxyproline relates to the collagen content and was measured with a chloramine-T assay as described by Huszar et al,18 (link) based on a standard curve of trans-4-hydroxyproline (Sigma).
7
Lanthanide-Labeled Protein Binding
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All reagents and solvents were of analytical reagent grade and were used without further purification. 5-hydroxyflavone, SmCl3·6H2O, EuCl3·6H2O, GdCl3·6H2O and TbCl3·6H2O, human transferrin and human serum albumin and double-stranded calf-thymus DNA were purchased from Sigma Aldrich Chemical Co. (Schnelldorf, Germany). All other chemicals and solvents were commercially available, and used without further purification.
8
Satellite Cell Proliferation Assay
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Satellite cell proliferation was determined by measuring the DNA concentration per well using Hoechst 33258 fluorochrome as previously described (Green and Sambrook, 2017 ). Cells were cultured in 24-well plates as described above. After 24 h of cell attachment in the plating medium, the medium was changed to growth medium, and 20S was added at 0.0 (control), 0.25, 0.5, and 1.0 μmol, and the cells were harvested at 0, 24, 48, and 72 h. For sample collection, 200 μL of 0.05% trypsin-EDTA (Gibco-Invitrogen, Carlsbad, CA) in the buffer (TNE: 10 mmol Tris, 2 mol NaCl, and 1 mM EDTA) was added to each well and incubated for 7 min, and then the plates were maintained at −70°C overnight. After thawing the plates at room temperature, the TNE buffer containing 0.2% (1 mg/mL) Hoechst dye (Sigma-Aldrich) was added to each well, and the plates were gently agitated for 1 to 2 h. DNA-incorporated Hoechst dye was measured using a Fluoroskan Ascent FL plate reader (Thermo Electron, Milford, MA). A standard curve with double-stranded calf thymus DNA (Sigma-Aldrich) was used to determine the sample DNA concentration. Two to 3 independent experiments were performed with 4 replicate wells per treatment condition.
9
Cell Proliferation Assay with Hoechst 33258
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Proliferation was assayed through measurement of DNA concentration using Hoechst 33258 fluorochrome as previously described (McFarland et al. 1995 ). Briefly, every 24 h, medium was removed, one plate was collected and washed with phosphate‐buffered saline (PBS: 137 mmol/L NaCl, 2.68 mmol/L KCl, 1.47 mmol/L KH2PO4, and 7.81 mmol/L Na2HPO4, pH 7.08) then stored at −70°C until all plates were collected. Plates were thawed for 15 min at room temperature and treated with 200 μL 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA; Invitrogen) in buffer containing 10 mmol/L Tris, 2 mol/L NaCl, and 1 mmol/L EDTA (TNE) for 7 min and returned to −70°C overnight. Each standard curve and cell culture well was treated with TNE buffer containing 0.2% (1 mg/mL) Hoechst dye (Sigma‐Aldrich) and allowed to shake for 1–2 h. The concentration of DNA labeled with Hoechst dye was measured on a Fluoroskan Ascent FL scanner (Thermo Fisher Scientific, Waltham, MA) and calculated based on a standard curve of double‐stranded calf thymus DNA (Sigma‐Aldrich) run in a plate previously trypsinized with the culture plates. The proliferation assay was plated in two separate experiments with four replicate wells per experiment.
10
Preparation and Characterization of PFB and TMB
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Double stranded calf thymus DNA (1 mg/mL) and single stranded calf thymus DNA (0.75 mg/mL) solutions were obtained from Sigma Chemical Company and prepared in 10 mM Tris-HCl buffer at pH 7.4 and stored at 8 °C. The stock solutions of 6 mM and 3 mM of both PFB and TMB were prepared in water/acetone (10% v/v) mixture and stored at room temperature in the dark. The colour of the fresh solution was bright yellow. These solutions remained stable for a few months. UV-Vis spectra and cyclic voltagrams were acquired to confirm the stability of these drug solutions.
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