BHK21, Vero, and HmLu-1 cell monolayers were tested for plaque formation. The cells were transferred to 6-well plates and incubated with 200 µL of viral inoculum for 1 h at 37 °C. After removing the inoculum, the cells were overlaid with DMEM supplemented with 2% FBS and 0.8% SeaKem GTG agarose (Lonza, Chiba, Japan). Subsequently, the cells were incubated at 37 °C for 3–7 days until the plaque grew to a countable size. Cells were fixed in formalin and stained with crystal violet for plaque visualization.
Seakem gtg agarose
Manufactured by Lonza
Sourced in Japan, Switzerland
SeaKem GTG agarose is a high-quality agarose product designed for use in gel electrophoresis applications. It is a natural polysaccharide extracted from red seaweed, offering consistent performance and reliable results. The product is suitable for a range of molecular biology techniques, including DNA and RNA separation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated peroxidase, by Thermo Fisher Scientific (2 mentions)
Biotinylated anti mouse igg antibody, by Merck Group (2 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Nusiev gtg agarose, by Lonza (2 mentions)
Superscript vilo kit, by Thermo Fisher Scientific (1 mentions)
Myecl imager, by Thermo Fisher Scientific (1 mentions)
Solidworks 2019, by Dassault Systèmes (1 mentions)
Formalin neutral buffer solution, by Fujifilm (1 mentions)
Seaplaque agarose, by Lonza (1 mentions)
Allprep mini kit, by Qiagen (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Proflex pcr system, by Thermo Fisher Scientific (1 mentions)
Fluoview fv300 clsm system, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Qubit dsdna broad range assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
T4 endonuclease 5, by New England Biolabs (1 mentions)
Sybr green 2, by Lonza (1 mentions)
16 protocols using seakem gtg agarose
1
Plaque Assay for Viral Quantification
2
Quantifying SORBS2 Isoform Expression
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Total RNA was collected using RNeasy (Qiagen) from WT and SORBS2 KO MDCKII cells grown on culture dishes until confluency. Reverse transcription was performed using SuperScript® VILO™ kit (ThermoFisher, Waltham, MA). qRT-PCR was performed as previously described [28 (link)] using primers for canine SORBS1, SORBS2, SORBS3 and ZO-1 (SORBS1 and SORBS3 primers: S2 Table , ZO-1 primers: previously published [28 (link)]).
For SORBS2 isoform identification, mRNA was collected from WT SKco15 and MDCKII cells and reverse transcription was performed as described above. Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers,S2 Table ) and DNA was amplified (Phusion, HF kit, New England Biolabs). The PCR products were loaded to a 1% agarose gel (SeaKem®GTG® agarose, Lonza) and separated by electrophoresis. Ethidium bromide-stained DNA bands were visualized by UV imaging (MyECL imager, ThermoFisher).
For SORBS2 isoform identification, mRNA was collected from WT SKco15 and MDCKII cells and reverse transcription was performed as described above. Primers were designed that could identify all, or specific isoforms, of SORBS2 in canine- or human cells (SORBS2 primers,
3
Fabrication of Agarose Microwell Plates
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Agarose microwell plates (61 wells) were fabricated using a 3D-printed mold and a polydimethylsiloxane (PDMS) mold, as illustrated in Supplementary Fig. S2 . First, a microwell plate was designed using SolidWorks 2019 (Dassault Systèmes SolidWorks Corporation). A 3D-printed mold of a microwell plate was made with a printer (QIDI TECH Shadow 5.5S printer), poly(ethylene glycol) diacrylate (PEGDA, Mn: 250) as a resin, 1%(w/w) photoinitiator (Omnirad 819), and 1% (w/w) photosensitizer (2-Isopropylthioxanthone). The mold was immersed in ca.100 % ethanol for more than 1 min and was exposed to UV light for 4 min, then heated at 80 °C overnight. The mold was coated with Parylene (DPXC, CAS No. 28804-46-8; Parylane Japan) using a PDS-2010 Labcoter 2 (Specialty Coating Systems Inc.). Parylene was used for making PDMS (Silpot184; Toray-Dow Corning) easy to peel off. A PDMS mold was fabricated by conventional soft lithography. PDMS (elastomer: curing agent = 10:1) was cast into the parylene-coated molds and heated to solidify them. The solidified object was taken off from the molds. Agarose (2%) (SeaKem GTG Agarose, Lonza) was poured into the PDMS mold. After solidifying the agarose, the agarose microwell plate was removed from the mold and then immersed in TS basal medium until use.
4
Plaque Assay for Virus Quantification
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The plaque assay was performed as described previously [31 (link)]. Briefly, confluent ST cells on a 12-well plate were inoculated with 0.1 mL each of 10-fold serially diluted viruses in MEM/BSA and incubated for 1 h at 37 °C. Cell were then washed with MEM/BSA, covered with 1 mL of MEM/BSA containing TPCK-trypsin (0.5 μg/mL) and 1% Seakem GTG agarose (Lonza Japan, Chiba, Japan), and incubated at 37 °C for 3 days. Subsequently, 30% formalin in PBS (0.5 mL) was added to each well to fix the cells at 4 °C overnight. After formalin and agarose were removed, the cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min at 23 °C. After blocking with BlockAce (KAC, Hyogo, Japan), the cells were incubated with anti-D/OK mouse immune serum for 60 min, biotinylated anti-mouse IgG antibody (#B7264, Sigma-Aldrich Japan, Tokyo, Japan) for 30 min, and then a complex with streptavidin (8 µg/mL) (Fujifilm Wako Chemicals, Miyazaki, Japan) and biotinylated peroxidase (4 µg/mL) (Invitrogen/Thermo Fisher Scientific, Tokyo, Japan) for 30 min. The plaques were visualized using a DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s instructions.
5
Multiplex PCR for Detecting Antibiotic Resistance Genes
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Isolates that tested positive for AmpC, ESBL, or carbapenemase phenotypes were subjected to PCR to confirm the genes encoding for AmpC (blaMOX-M, blaACC-M, blaEBC-M, blaFOX-M, blaCIT-M and blaDHA-M) [47 (link)], ESBLs (blaCTX-M) [48 (link)], carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP and blaOXA-48) [49 (link)], and other β-lactamase genes (blaTEM and blaSHV) [48 (link)] with slight modifications. Crude DNA (10 µL) was extracted from pure overnight cultures and suspended in 200μL of molecular-grade nuclease-free water, heated for 10 min at 98 °C, and centrifuged for 5 min at 4 °C and 20,000 g, as previously suggested by Quansah [42 (link)]. The supernatant was transferred into sterile 1.5 mL Eppendorf® tubes and used as a template for the PCR. For the PCR amplification, each reaction mix of 25 µL consisted of 12.5 uL of Green PCR Master Mix (2×) (DreamTaq, Thermo Scientific, Waltham, MA, USA), 4.5 µL of primer mix, 6 µL of molecular-grade nuclease-free water and 2 µL of crude DNA template, as previously demonstrated by Khurana et al., 2018 [49 (link)]. The primers used for PCR amplification and cycling conditions are listed in Table 4 . All PCR amplicons were analyzed via horizontal gel-electrophoresis in a 2% (weight/volume) agarose gel (SeaKem®GTG®Agarose, Lonza, Basel, Switzerland) using Tris/Acetate/EDTA 50× concentrate buffer.
6
Viral Plaque Assay Protocol
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Viruses were serially diluted 10-fold in growth medium. Confluent monolayers of VeroE6/TMPRSS2 cells on 12-well cell culture plates (AGC TECHNO GLASS Co., Ltd., Tokyo, Japan) were washed twice with growth medium and infected with 100 μL of virus diluted from 100 to 106 and were incubated for 60 min at 37 °C. After the virus inoculum was removed, the cells were washed with growth medium and overlayed with a 1:1 mixture of 2× growth medium and 2% agarose [SeaKem® GTG™ agarose (Lonza, Basel, Switzerland) and SeaPlaque™ agarose (Lonza) in 1:1]. The cells and virus were then incubated at 37 °C for 48 h, fixed with 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and the plaques formed by the viruses were counted.
7
Nuclear and Mitochondrial DNA Isolation
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Total human DNA (consisting of nuclear DNA and mtDNA) was isolated from human iPS cells (clone A4 established in our lab) using Allprep Mini Kit (QIAGEN). Total mouse DNA was isolated from C57BL/6 mouse liver (Charles River) using DNeasy Blood and Tissue Kit (Qiagen). The NCAs were prepared from total DNA using Q5 High-Fidelity DNA Polymerase (New England Biolabs) by 30-cycle amplification (primers and PCR conditions are shown in S1 Fig ) [24 (link)]. Single-band amplicons (9.2 and 11.2 kbp for human, 8.0 and 8.6 kbp for mouse) were excised from agarose gel after electrophoresis (0.7% SeaKem GTG agarose; Lonza) and purified using QIAquick gel extraction kit (QIAGEN) (S1 Fig ).
8
Spheroid Formation from Single Cells
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Cell suspension were diluted to 100 cells in 20 mL of RPMI-medium and then seeded onto 96-well-plates (200 µL per well), which were previously coated with an 0.7% agarose (SeaKem GTG Agarose, Lonza Group, Basel, Switzerland) in PBS solution, in order to inhibit adhesion and therefore guarantee spheroid forming starting at a single-cell level. The formed spheroids were counted after 15 days.
9
Genotypic Characterization of Bacillus thuringiensis Isolates
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Bt isolates were genotypically characterized according to the method detailed in S1 Table . The presence of the cytK1/2, ces, hlyII, nheA/B/C and hblA/C/D genes was determined by PCR with the ProFlex PCR System (Applied Biosystems), using respective primers and methods (S1 Table ). To assign Bt isolates to one of the seven described phylogenetic groups, a part of the panC gene was amplified as described (S1 Table ) and sequenced (Eurofins MWG Operon). For publicly available sequences, the full sequence of panC was extracted with BLAST [31 (link)], using the ATCC 14579 panC sequence (AE016877.1) as the query. The phylogenetic classification was then performed online (https://www.tools.symprevius.org/Bcereus/ ) using a public algorithm based on panC sequence similarity and the statistical significance of matches with database sequences [9 (link)]. All the PCR products were analyzed by electrophoresis onto 2% agarose gels (made with a 50/50 mix of Seakem® GTG™ Agarose and NuSiev® GTG™ Agarose, Lonza), with a run in 1X TBE, for 1.5 hours at 90V.
10
Molecular Diversity Profiling of Bacillus cereus
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To study the diversity of Bc isolates, we used a coliphage M13 sequence-based PCR (M13-PCR) derived from a Random Amplified Polymorphic DNA (RAPD) technique and adapted from [32 (link)], according to the method described in S1 Table . The PCR products were separated by electrophoresis onto 1% agarose gels (made with a 50/50 mix of Seakem® GTG™ Agarose and NuSiev® GTG™ Agarose, Lonza), with a run in 1X TBE, for 10 min at 50V, followed by 3.5h at 90V. Gels were stained with ethidium bromide. For this study, the electrophoretic patterns of 59 representative FBO-Bt and 19 representative insecticide Bt isolates were analyzed and compared using Bionumerics 7.6. A dendrogram was constructed based on pairwise Dice similarity coefficients calculations [33 ] and UPGMA clustering, with tolerance and optimisation set at 1%. The Bt isolates were tested three times by M13-PCR typing and similar visual patterns were observed.
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