The contractile activity of NHCFs was measured by collagen gel contraction assay. After the knockdown, 2 × 105 cells were resuspended with serum-free media containing 1 mg/mL Collagen type I (#354231, Corning Life Sciences, NY, USA). The collagen–cell mixture was mixed with 1M NaOH and immediately transferred to the wells of a 12-well plate. The gels were allowed to polymerize at room temperature for 30 min and gently detached with a sterilized spatula. To initiate contraction, the gels were covered with serum-free media containing TGF-β1. The gels were imaged at 0, 24, and 48 h by use of the ENDURO GDS gel documentation system (Labnet International, Iselin, NJ, USA), and gel size was calculated by using ImageJ software.
Enduro gds gel documentation system
Manufactured by Labnet
Sourced in United States
The ENDURO GDS Gel Documentation System is a compact and versatile imaging device designed for the visualization and documentation of DNA and protein gels. It utilizes a high-resolution camera and specialized software to capture and analyze gel images.
Automatically generated - may contain errors
Lab products found in correlation
Collagen type 1, by Corning (1 mentions)
Proflex pcr system, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Chelex 100, by Bio-Rad (1 mentions)
Hotstartaq pcr, by Qiagen (1 mentions)
Go taq flexi dna polymerase, by Thermo Fisher Scientific (1 mentions)
Proflex pcr system thermocycler, by Thermo Fisher Scientific (1 mentions)
Sanger method, by Macrogen (1 mentions)
5 protocols using enduro gds gel documentation system
1
Collagen Gel Contraction Assay for NHCFs
2
Profiling Campylobacter Virulence Genes
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Six virulence genes; Cytolethal distending toxin subunits; cdtA, cdtB, and cdtC, Campylobacter adhesion to fibronectin protein (CadF), phospholipase A (pldA), and Campylobacter invasion antigen B (ciaB) were screened from Campylobacter jejuni isolates. The primers and PCR cycling conditions were obtained from the previously published articles [10 (link), 38 (link), 39 (link)] and are shown in Table 2 . The 25 μL total reaction consisted of 12.5 μL of a 2X DreamTag Green Master Mix (0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, and 0.4 mM dTTP, 4 Mm MgCl2, and loading buffer), 8.5 μL of nuclease-free water, 2.0 μL of template DNA, and 1.0 μL of each oligonucleotide primer. PCR reactions were performed using the ProFlex PCR System (Applied Biosystems, USA). Amplified PCR products were resolved on a 1% (w/v) agarose gel in a 40 mM Tris (Sigma Aldrich, US), 1X TAE buffer (20 mM acetic acid (Merck, US), and 1 mM EDTA (Merck, South Africa) at pH 8.0), stained with 0.001 μg/mL ethidium bromide and visualized under ultraviolet (UV) light using the ENDURO GDS Gel Documentation System (Labnet International Inc., US).
3
Screening Bacterial PHA Production via Nile Blue
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All of 110 bacterial isolates were tested for PHA production using Nile Blue A staining method. The bacteria isolates were cultivated on agar plates containing MB and the selected sugar (2% w/v) and incubated for four days at 32°C. Nile Blue A (0.5 𝜇g/mL) was directly added to a rich Marine Broth agar medium; thus, the bacterial cells were grown in the presence of the dye. Subsequently, the bacteria were exposed to UV illumination (320 nm) using the ENDURO™ GDS Gel Documentation System (Labnet International, Inc. Israel). This technique allowed rapid screening of the viable colonies for PHA production and considered to be a powerful tool for distinguishing between PHAnegative and PHA-positive strains. The bacteria that have shown a bright white fluorescence on irradiation with UV light were selected as potential PHA accumulators. The selected bacteria were repeatedly grown on different sugars in Marine Broth plates, and the accumulation of PHA on each sugar was also examined by Nile Blue staining. All experiments were carried out in triplicates.
4
DNA Extraction and PCR Identification of Resistant Nematodes
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For DNA extraction, the Chelex-100 (Bio-Rad Laboratories, CA, USA) procedure was used [21 ]. In a DNA vial containing ~100 larvae of GINs from sheep, 30 μL of Chelex was added, followed by 5 μL of Proteinase K. The vial was vortexed briefly and centrifuged at 1,975× g for 1 min. All DNA samples were stored at −20°C for future use.
Conventional PCR targeting the internal transcribed spacer 2 gene with genus-specific primer pairs (Table-1 ) [27 (link), 28 ] was used to detect resistant nematodes, namely, Haemonchus spp., Oesophagostomum spp., Trichostrongylus spp., and Teladorsagia spp. The PCR assays were conducted in a 25 μL reaction volume, using 12.5 μL HotStar Taq PCR (Qiagen, Valencia, CA) master-mix, 8.5 μL double distilled water, 1 μL forward primer, 1 μL of reverse primer, and 2 μL DNA template. The PCR reaction was conducted under the following conditions: initial denaturation at 95°C for 15 min; 50 cycles of denaturation at 94°C for 1 min (annealing temperatures are shown in Table-1 ) and extension at 72°C for 1 min; and final elongation at 72°C for 1 min. Then, 5 μL amplicon was resolved by gel electrophoresis using 1% (w/v) agarose gel stained with ethidium bromide (0.1 μg/mL) and visualized under ultraviolet light using the ENDURO GDS Gel Documentation System (Labnet International Inc., US).
Conventional PCR targeting the internal transcribed spacer 2 gene with genus-specific primer pairs (
5
PCR Amplification and Sanger Sequencing
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For the PCR reaction, a ProFlex PCR System thermocycler (Applied Biosystems, MA, USA) and GoTaq® Flexi DNA polymerase were used (Madison, USA), following the manufacturer's guidelines. The amplification products were revealed by horizontal electrophoresis using a subcell electrophoresis chamber (Bio-Rad, CA, USA) on a 2% agarose gel stained with HydraGreen™ (ACTGene, NJ, USA) and a molecular weight marker of 100 bp (New England Biolabs, USA). The gel was visualized and documented using the ENDURO GDS Gel Documentation System (Labnet International, NJ, USA). The amplification products were purified and sequenced by the Sanger method (Macrogen Inc., Korea).
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