Before CCI injury, each animal was anesthetized with 5% isoflurane and buprenorphine was injected subcutaneously (0.3 mg/ml, 0.05 ml/300 g; Henry Schein). Animals were placed on a stereotaxic frame attached to a temperature-controlled heating pad (37°C) with their scalp shaved and sanitized (70% EtOH and 3% povidone-iodine). Following a sagittal incision, the periosteum was cleaned using Etch Gel (Phosphoric Acid Etching, Henry Schein), and a craniotomy was performed using a 5-mm-diameter trephine bur fitted to an electronic drill. A 3-mm CCI tip was fitted onto the pneumatic piston and positioned in contact with the surface of the dura (fully extended position) and then retracted to adjust for an impact depth of 2 mm. A severe CCI injury was induced by programming the piston speed to 2.25 m/s and a dwell time of 250 ms, resulting in an initial 3-mm-diameter injury with a depth of 2 mm. Absorbable gelatin (Gelfoam, Pfizer) was applied to the injury site, and sterile cotton swabs were used to remove excess blood. The Gelfoam was then removed, and the injury site was covered completely with a layer of 1% sterile SeaKem (Lonza) agarose. Skin flaps were sutured together, closing the wound. Triple antibiotic cream was applied on the sutured skin.
Seakem
Manufactured by Lonza
Sourced in United States
SeaKem is a laboratory equipment product manufactured by Lonza. It is used for agarose gel electrophoresis, a widely used technique in molecular biology and genetics for the separation and analysis of DNA and RNA molecules. The core function of SeaKem is to provide a reliable and consistent platform for performing these electrophoresis experiments.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Gelfoam, by Pfizer (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Qiagen (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Veriti, by Thermo Fisher Scientific (1 mentions)
G box imaging system, by Syngene (1 mentions)
On column dnase treatment, by Qiagen (1 mentions)
Oligo dt, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Gotaq polymerase, by Promega (1 mentions)
Cm1850, by Leica (1 mentions)
Seaplaque, by Lonza (1 mentions)
Gelred stain, by Biotium (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Maldi tof ms analysis, by Bruker (1 mentions)
Chromatography grade ethanol, by Merck Group (1 mentions)
Nucleospin tissue extraction kit, by Macherey-Nagel (1 mentions)
13 protocols using seakem
1
Controlled Cortical Impact Injury Model
2
Genomic DNA Extraction and PCR Amplification of Virulence Genes
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Total genomic DNA was extracted from overnight cultures grown in tryptone soy broth (TSB) (Oxoid) at 37°C using the protocol for Gram-negative bacteria in the DNeasy Blood and Tissue kit (Qiagen, Oslo, Norway). All PCR reactions were performed with 25 μl reactions containing 1x PCR buffer (1.5 mM MgCl2), 200 μM of each nucleotide, 0.2 μM each primer, 2.5 U Taq polymerase (Qiagen), and 50–100 ng template DNA. The PCR amplification cycles were as follows: Initial denaturation at 95°C for 15 min, 30 cycles of denaturation at 95°C for 30 s, annealing for 30 s at temperature given in Table 1 , and extension at 72°C for 60 s, followed by a final extension at 72°C for 7 min. All PCR reactions were performed in duplicate in separate experiments. PCR products were visualized by electrophoresis in a 1.5% agarose gel (SeaKem, Lonza Group Ltd., Basel, Switzerland) in 1x TAE buffer. Two random PCR products of each detected virulence gene (ast, act, alt, aerA, and hlyA) were excised from the agarose gel and purified using the GeneJET Gel Extraction Kit (Thermo Scientific, Oslo, Norway) and sequenced (Eurofins Genomics, Ebersberg, Germany) to confirm the amplicon specificity.
3
Genomic DNA Isolation and PCR Amplification
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Genomic DNA was isolated from the leaf samples by following a standard protocol used for plants [27 ]. The isolated DNA samples were checked for their quality and quantity by using agarose gel electrophoresis (0.8% agarose gel) and spectrophotometer (Thermo electronic corporation UV1) respectively and then were used to set the PCR reactions [35 (link)]. The PCR reaction mixture contained 50ng template DNA, 5 picoM each of forward and reverse primers, 200μM dNTPs, 1X PCR buffer (10mM Tris-HCl, pH 8.3, 50mM KCl, 1.5mM MgCl2 and 0.01mg/ml gelatin) and 0.5U of Taq DNA polymerase (JONAKI) in a reaction volume of 10μl. Amplification cycling was performed in a gradient programmable master cycler (Veriti, Applied Biosystems). The PCR condition was with one cycle of denaturation at 95°C for 5 min, followed by 35 cycles at 95°C for 45s, 55°C for 45s, and 72°C for 1 min, and with a final extension at 72°C for 10 minutes. The final PCR products were resolved by electrophoresis on 3% agarose (Sea Kem, Lonza) gel and documentation was done using gel documentation system (BIO- RAD), images were stored for further scoring and permanent records.
4
Quantifying Relative Transcript Levels
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Total RNAs were prepared using an RNeasy Plant Mini kit with on-column DNase treatment (Qiagen), and cDNAs were synthesized using a RevertAid H Minus First Strand cDNA Synthesis kit with Oligo dT (Thermo Scientific). Steady-state transcript levels were determined following a robust reverse transcription (RT)-PCR procedure (see Supplementary Table S2 available at JXB online for primer sequences). Briefly, after PCR amplification with GoTaq polymerase (Promega), PCR products were separated in a 1.5 % LE agarose gel (SeaKem; Lonza) in sodium borate buffer (Brody et al., 2004 (link)). As an inter-run calibrator, a common DNA fragment was loaded on each gel, enabling gel-to-gel comparisons. After gel staining with GelRed (Biotium), the gels were imaged with identical settings using a G:BOX imaging system (Syngene), and absolute band intensities were determined in Photoshop (Adobe Systems Software). Expression values were normalized to EF1α and the inter-run calibrator. Relative AtFRD3S steady-levels were calculated as a percentage of total AtFRD3 transcripts using the following formula: AtFRD3S/(AtFRD3S+AtFRD3L)×100.
5
Spreading Assay for Bacterial Growth
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For spreading assays, TSA containing 0.25 (TSA-0.25) or 0.4% (TSA-0.4) agarose (Seakem, Lonza Rockland, Inc.) was used. After autoclaving, medium was placed in a water bath at 55°C for 30 min. Plates containing 25 ml TSA-0.25 or TSA-0.4 were prepared and were then incubated at room temperature for 20 min before inoculation. Each plate was inoculated with 2 μl (1 × 107 CFU) of an overnight culture and was then allowed to dry for 15 min in a laminar flow hood. The plates were incubated at 37°C for 24 h to allow bacteria to spread. To determine the presence of water within colonies, the TSA-0.4 plates were tilted at a 30° angle during incubation according to the method described previously (Lin et al., 2016 (link)).
6
Agarose-Hyaluronic Acid Bioprinting
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Agarose support gels were made as previously described (56 ), with some modifications. Briefly, a 0.5 wt % agarose (SeaKem, Lonza) solution was mixed and autoclaved to melt and sterilize and then cooled while stirring at 700 rpm to form microgels. This microgel solution was then diluted in an 8 wt % solution of HA to give a final agarose concentration of 0.25 wt % and HA concentration of 4 wt %. Agarose was diluted to give a support gel that was soft enough to yield with extremely soft fiber solutions. HA was added to diluted agarose microgel solution to increase the viscosity and stabilize the printed structures while moving. Fiber solutions were extruded using 27-gauge needles and a submicroliter injection needle (World Precision Instruments, Nano-Fil 100), with two different extrusion printers (Allevi 2 and Velleman K8200), and photocrosslinked (400 to 500 nm, 5 mW/cm2 for 5 min) after printing. Filaments were imaged directly after printing and after releasing prints from the support bath at ~20 hours of culture by diluting with an equal volume of MSC medium.
7
3D Cell Culture Cryosectioning and Staining
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The 3D cell cultures were included in 2% agarose gel (Lonza Seakem, LE agarose cod.n.50004) just 1 h after the end of the experiment to obtain a macroscopic block suitable for slicing. After agarose inclusion, the samples were fixed by cooling at −20 °C and then sliced using a cryomicrotome (Leica CM 1850). The slices were deposed on a microscopy glass and properly stained. A sample of the matrix without cells was treated in the same way for comparison.
8
Genomic Fingerprinting via Rep-PCR Analysis
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To obtain the genomic fingerprints, the second typing method rep-PCR was performed using three primers: (GTG)5, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) for the analysis of 30 colonies for each experiment. The PCR procedure followed has been described previously [28 ]. Each reaction included 2 µL of DNA template, 1 µL (10 pmol/µL) of the reverse primer, 1 µL (10 pmol/µL) of the forward primer, and 16 µL of autoclaved distilled water to bring the total volume to 20 µL. The PCR products (5 µL) were then examined using 1.5% (w/v) agarose (Seakem, Lonza, Alpharetta, GA, USA) gel electrophoresis at 70 V for 5 h. The PCR amplification conditions for all the three rep-primers used in the present study are described in Table S1 .
9
Quantifying Infectious Virus and Bacteriophage
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To quantify infectious virus from CM experiments, plaque assays were performed. For MNV-1, RAW 264.7 cells were seeded into six-well plates and incubated overnight at 37 °C and 5% CO2. Samples were serially diluted in complete DMEM, plated in duplicate wells, and plates incubated for 1 h at room temperature while rocking. The inoculum was then aspirated, and the cells overlaid with 1.5% SeaPlaque (Lonza) agarose in MEM supplemented with 10% FBS. Plates were incubated at 37 °C and 5% CO2 for 3 d. Plaques were visualized by overlaying with 2 mL of 1.5% SeaKem (Lonza) agarose containing 1% neutral red per well for 5 h [54 (link)].
Infectious titer of bacteriophage MS2 was quantified with a double-layer agar method using the E. coli strain C3000 (ATCC 15597) as host [47 (link)]. Following the treatment with chitosan microparticles, 700 µL of the serially diluted MS2 were mixed with 300 µL of log-phase growing E. coli and 8 mL top agar (tryptic soy broth with 0.6% agar), then overlaid on a bottom agar (tryptic soy broth with 1.2% agar). Plaque forming units (PFU) were counted following 24 h incubation at 37 °C.
Infectious titer of bacteriophage MS2 was quantified with a double-layer agar method using the E. coli strain C3000 (ATCC 15597) as host [47 (link)]. Following the treatment with chitosan microparticles, 700 µL of the serially diluted MS2 were mixed with 300 µL of log-phase growing E. coli and 8 mL top agar (tryptic soy broth with 0.6% agar), then overlaid on a bottom agar (tryptic soy broth with 1.2% agar). Plaque forming units (PFU) were counted following 24 h incubation at 37 °C.
10
Agarose Gel Electrophoresis of cirDNA and PCR Products
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Agarose gels were made up with 1% agarose (Lonza Seakem, Cat# 50002) in 40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.4 buffer (TAE buffer), plus 10 μL Gel Red stain (Biotium, Cat# 41003) per 100 mL added just before the gel was poured. For gels visualising cirDNA, 0.1 μL of 100 bp DNA ladder MWM (molecular weight markers) (New England Biolabs, Cat# N3231S) was loaded, alongside the sample volumes indicated in the figure. For gels visualising PCR product 1 μL of DNA ladder MWM was loaded alongside 4 μL of control untreated PCR product and 25 μL of bis-treated PCR product, that had been obtained from 40 μl of untreated PCR product. Gels were run at 100 V for the times indicated in the figure legends.
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