Cells were lifted immediately following mock or 200 μM H2O2 treatment and 103 cells were mixed with 0.5% low-melting point agarose (Bio-Rad) (37 °C in 1× TBE). The cell suspension was spread onto a comet slide (Trevigen) and immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH10), 1% Triton X-100) for 30 min at 4 °C. Slides were immersed in Alkaline Comet Buffer (300 mM NaOH, 1 mM EDTA) for 30 min before electrophoresis in same buffer at 1 Volt/cm (∼300 mA) for 30 min. Slides were then washed in dH2O and dried at 45 °C for 30 min. The DNA was stained using SYBR® Green I (Sigma) (1:10,000) before being dried completely and visualised using a Nikon Eclipse Ti microscope. Quantitation of comet tail moments was performed on a minimum of 50 cells using ImageJ plugin where the densitometry of the head and tail, as well as length were measured to calculate the comet tail moment. Assays were performed using at least three biological repeats. Results are displayed as mean ± S.D.
Low melting point agarose
Manufactured by Bio-Rad
Sourced in United States, Germany, France
Low-melting point agarose is a type of agarose, a polysaccharide derived from red seaweed, used in various biotechnology applications. It is characterized by a lower melting temperature compared to standard agarose, allowing for gentler handling and processing of heat-sensitive samples.
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Lab products found in correlation
Normal melting point agarose, by Bio-Rad (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Ethidium bromide, by Bio-Rad (3 mentions)
Penicillin streptomycin, by Lonza (3 mentions)
Trypsin edta, by Lonza (2 mentions)
Chef mapper system, by Bio-Rad (2 mentions)
Acrylamide, by Bio-Rad (2 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions)
Methylcellulose m3234, by STEMCELL (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Acetic acid, by Avantor (2 mentions)
Sybr green 1, by Merck Group (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Lysostaphin, by Merck Group (1 mentions)
Proteinase k, by NZYTech (1 mentions)
Chef dr 3, by Bio-Rad (1 mentions)
Thermal imaging system fti 500, by GE Healthcare (1 mentions)
Lysozyme, by Merck Group (1 mentions)
24 protocols using low melting point agarose
1
Comet Assay for DNA Damage
2
Molecular Fingerprinting of Staphylococci by PFGE
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Molecular fingerprinting of staphylococci was performed by PFGE using a CHEF-DRIII apparatus (Bio-Rad Laboratories, San Diego, USA). Bacterial cultures were grown overnight on Columbia agar supplemented with 5 % sheep blood (BioMérieux) and a cellular suspension of 5 × 109 CFU/mL incorporated into 1.5 % low melting point agarose (BioRad). Discs were immersed into a lysis solution with lysostaphin (Sigma-Aldrich) (50 μg/ml), lysozyme (Merck) (100 μg/ml) and RNase (Roche) (50 μg/ml) at 37 °C for 3 h. After lysis, discs were incubated with proteinase K (NZYTech, Portugal) (1 mg/ml) for 17 h at 50 °C, followed by overnight digestion with SmaI (Takara) at 25 °C. Digested DNA was submitted to electrophoresis in 1 % agarose gel (Seakem LE) for 23 h at 14 °C and 6 V/cm with pulse times of five to 35 s. Lambda Ladder PFG Marker (BioLabs) 50 μg/ml was used as molecular weight marker. Agarose gels were stained with ethidium bromide and visualized by transillumination under UV (Pharmacia Biotech, Thermal Imaging System FTI- 500). BioNumerics 7.5 software (Applied Maths, Kortrijk, Belgium) was used to register macrorestriction patterns and clustering analysis was performed using DICE similarity coefficient and the unweighted-pair group method with arithmetic mean (UPGMA).
3
Phage genome size estimation by PFGE
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Pulsed field gel electrophoresis (PFGE) was conducted as described by Lingohr et al. (2009 (link)). A CsCl purified phage suspension was mixed with an equal volume of low-melting point agarose (Bio-Rad Laboratories, Hercules, USA). Prepared plugs were placed in lysis buffer (50 mM Tris, 50 mM EDTA, 1% SDS) and digested with proteinase K for 2 h at 54°C. Plugs were then washed twice with TE buffer (10 mM Tris, 1 mM EDTA [pH8.0]) and subjected to electrophoresis on a 1% agarose gel using Bio-Rad CHEF-DR® II PFGE apparatus (Bio-Rad Laboratories, Hercules, USA) at 6 V/cm (200 V) with 60–120 switch time ramp for 24 h. Yeast chromosome PFGE markers (Bio-Rad Laboratories, Hercules, USA) were used to allow estimation of phage genome size.
4
Autophagy-related protein quantification
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Dulbecco’s modified Eagle’s medium (DMEM) 4.5 g/L Glucose and fetal bovine serum (FBS) were from Dominique Dutscher (Brumath, France), Glutamax was from Life Technologies (Milano, Italy), trypsin-EDTA and penicillin/streptomycin were from Biowhittaker (Verviers, Belgium). Triton X-100, N-lauryl sarcosine, MD, Bafilomycin A1, and E64d-protease inhibitors were from SIGMA (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), NaOH, and Na2EDTA were from Baker (Deventer, The Netherlands). Tris and NaCl were from Carlo Erba (Milan, Italy). Normal-melting-point agarose, low-melting-point agarose, and ethidium bromide were from Bio-Rad Laboratories (GmbH, Munich, Germany). Nitrocellulose membranes for Western blotting were AmershamProtran™ 0.2 µm NC, Chicago, IL, USA. Antibodies used are rabbit anti-LC3 (#L7543, Sigma Aldrich, St. Louis, MO, USA), anti-ATG5 (A0731, Sigma Aldrich), Anti-ATG7 (clone D12B11, Cell signaling Technology, Danvers, MA, USA), and horseradish peroxidase-conjugated secondary antibodies from Jackson Immunoresearch. NP-40 was from Fisher Scientific (Illkirch, France). Western blots were revealed with Clarity Western ECL (Bio-Rad laboratories, Marnes-la-Coquette, France). Chemiluminescence was detected with a G:Box imaging system (Syngene, Cambridge, UK).
5
Quantifying Viral Infectivity in Vero Cells
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Ten-fold serial dilutions of virus samples were prepared, and 200 μl of each was used to inoculate Vero cells grown in six-well plates by incubation for 1 hour. Cells were then overlaid with DMEM containing 0.5% low–melting point agarose (Bio-Rad, USA) and 2% FBS. At 72 hpi, cells were fixed with 4% PBS-buffered formaldehyde for 1 hour, stained with 0.1% crystal violet for 30 min, and washed with water.
6
Reagents for Cell Culture Experiments
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Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), L-glutamine, trypsin EDTA and penicillin/streptomycin were from Biowhittaker (Verviers, Belgium). Triton X-100, N-lauryl sarcosine and menadione were from Sigma (St. Louis, MO). Dimethyl sulfoxide, NaOH and Na2EDTA were from Baker (Deventer, The Netherlands). Tris and trypan blue were from BDH (Poole, England); NaCl was from Carlo Erba (Milan, Italy). Normal-melting-point agarose, low-melting-point agarose and ethidium bromide were from Bio-Rad Laboratories (GmbH, Munich, Germany). Ribospin kit and Riboclear plus were purchased by GeneAll Biotechnology CO, Ltd (Seoul, Korea). cDNA was obtained via reverse transcription (by OriGeneTechnologies, Inc. (Rockville, MD, USA). SensiFast SYBR-based kit was from Bioline (London, UK). Custom primers were synthetized by IDT Integrated DNA Technologies, Inc. (Coralville, IA, USA).
7
Protelomerase-Based DNA Assembly Protocol
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TelN protelomerase, β-Agarase I, SbfI, MluI, NotI and 2 × NEBuilder® HiFi DNA Assembly Master Mix were purchased from NEB (Ipswich, MA, USA). The zymolysase-20 T was bought from Nacalai Tesque (Kyoto, Japan). The Low Melting Point Agarose, Micropulser and CHEF Mapper XA System were purchased from Bio-Rad (Hercules, USA). Dulbecco’s modified Eagle’s medium (DMEM) was bought from Corning (NY, USA) and the 10% fetal bovine serum was bought from BI (Kibbutz Beit-Haemek, Israel). The phenylmethyl sulphonyl fluoride (PMSF), Proteinase K, Kanamycin, Spectinomycin and GlycoBlue™ Coprecipitant were purchased from Sangon Biotech (Shanghai, China). The Azure Biosystems C150 was bought from Azure Biosystems (Dublin, CA, USA). The bacterial genomic segments for targeted cloning were from E. coli str. MG1655. The E. coli str. GB05RedTrfA (DH10B, fhuA::IS2, ΔybcC, ΔrecET, PRhaSR-γβαA, PBAD-trfA) was obtained from the laboratory of Professor Youming Zhang of Shandong University [29] (link). The tos sequence (519 bp) and TelN protelomerase expression gene (1896 bp), primers were synthesized from GENEWIZ (Suzhou, China) (Supplementary Table S1 and Table S2).
8
Clonality Assessment of cfr-positive Isolates
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Pulsed-field gel electrophoresis (PFGE) was used to determine the clonality of the cfr-positive S. sciuri isolates. Whole-cells were packed with an equal volume of 2% (wt/vol) low melting point agarose (Bio-Rad, Laboratories, Hercules, CA, USA) dissolved in EC buffer (Liu et al., 2014 (link)) and poured into plug molds. The frozen plugs were addressed according to the previously described with minor modification. The digested fragments were separated using a CHEF-DRIII system (Bio-Rad) with a clamped homogeneous electric field of 6 V/cm, using a 120° switch angle for 24 h at 14°C, with the pulse time linearly ramped from 3 to 40 s. PFGE was performed for all cfr-positive E. coli isolates using the CHEF Mapper System (Bio-Rad), according to a previously described protocol (Chen et al., 2007 (link)). Comparison of PFGE patterns was performed using the BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) using the Dice coefficient (1.5% optimization and 1.0% tolerance); a similarity cutoff of 80% was used to identify a PFGE cluster.
9
3D Light Sheet Imaging of Organoids
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Whole-mount immunostained organoids were embedded in an embedding solution as previously described (Dekkers et al., 2019 (link)) with light-sheet glass capillaries. Briefly, 0.4 g of low-melting point agarose (Bio-Rad, 1613111) was fully dissolved in 10 ml of water and 10 ml of fructose-glycerol clearing solution added and mixed well to obtain a clear solution. A Zeiss Z1 light sheet microscope was used for imaging. Samples were imaged by placing the sample-containing capillary in the light sheet chamber filled with fructose-glycerol clearing solution and pushing down the embedded sample from the capillary to be exposed for imaging. A 20× detection objective (clearing immersion N.A.=1.0) was used to acquire whole z stack images with 1024×1024 frame size. Arivis Vision4D was used to process the z planes and generate 3D-rendering images that exported as .tiff files.
10
Protein Profiling of Cultivation Samples
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A quantity of 100 μg of proteins isolated after 7 and 18 h of cultivation was concentrated using Concentrator 5301 (Eppendorf, France) at room temperature. Samples were mixed with rehydration buffer containing 6 M urea, 2 M thiourea, 4% CHAPS, 0,4% dithiothreitol (DTT), and 2% Bio-Lyse 3/10 Ampholyte (Bio-Rad) and few grains of bromophenol blue (BB). Proteins were then loaded into 17 cm IPG strips (pH 4-7, Bio-Rad) by active rehydration at 50 V for 12 h. After that, the isoelectric focusing (IEF) was performed using the Bio-Rad IEF program as follows: from 50 to 250 V for 3 h, from 250 to 6,000 V for 3 h and at 6,000 V until reaching 54,000 Vh. Strips were then equilibrated in migration buffer containing 6 M urea, 50 mM Tris-HCl (pH 8.8), 2% SDS, 30% glycerol, 2% DTT, 4% iodoacetamide, and few grains of BB. The second dimension was performed using SDS-PAGE with 12% acrylamide gels. The IPG strips were immobilized to the gel using 1% low-melting point agarose (Bio-Rad). The migration ran at 40 mA/gel using Protean II xi cell (Bio-Rad) at 14°C, until the bromophenol blue reached the base of the gels. Proteins in gels were finally silver stained and scanned with a GS-800 densitometer operated with the QuantityOne® software (Bio-Rad). Three independent experiments were performed, with two technical replicates for each of them.
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