Viral titers were determined by plaque assay using an agarose and neutral red assay (previously described in reference 43 (link)). Briefly, Vero E6 cells were seeded at a density of 9 × 105 cells per well in 6-well tissue culture plates in normal growth medium with 10% heat-inactivated FCS. The next day, serial dilutions of virus were prepared in normal growth medium containing 2% FCS, media were removed from plates, and 400 µl of each dilution was added to the corresponding well. The plates were incubated for 1 h at 37°C with 5% CO2 with constant rocking. After incubation, media were removed from the wells, and a 2-ml primary overlay was added. The primary overlay consisted of 1% Seakem agarose (Lonza) mixed 1:1 with 2× Eagle’s MEM (EMEM; Lonza) containing 4 mM l -glutamine, 2 mM sodium pyruvate, and 4% FCS. Cells were incubated at 37°C with 5% CO2 for 7 days. On day 7, 2 ml of secondary overlay was added to each well; the secondary overlay consisted of 1% Seakem agarose (Lonza) mixed 1:1 with 2× EMEM (Lonza) containing 4 mM l -glutamine, 2 mM sodium pyruvate, 4% FCS, and 8% neutral red solution (Gibco). Cells were incubated at 37°C with 5% CO2 for 1 day, and then cell monolayers were inspected for plaques to determine a final titer.
Seakem agarose
Manufactured by Lonza
Sourced in United States, Switzerland
SeaKem agarose is a laboratory-grade agarose product manufactured by Lonza. It is a purified polysaccharide derived from red seaweed, commonly used as a gelling agent in various applications, such as gel electrophoresis and cell culture. SeaKem agarose provides a consistent, high-quality matrix for the separation and analysis of biomolecules.
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Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
Gotaq flexi dna polymerase, by Promega (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Quant ittm picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Agarose l03, by Takara Bio (1 mentions)
Vero e6 cells, by Corning (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
Crystal violet solution, by Merck Group (1 mentions)
Carboxymethylcellulose cmc, by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Gelred, by Biotium (1 mentions)
Horseradish peroxidase conjugated polyclonal rabbit anti human vwf, by Agilent Technologies (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
28 protocols using seakem agarose
1
Viral Titer Quantification by Plaque Assay
2
Soft Agar Assay for HCT116 Cells
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HCT116 or its clonal derivatives were mixed with 1 mL of 0.37% top agar (SeaKem agarose, Lonza) in M25 (McCoy's 5A medium supplemented with 25% FBS) plus 1× Antibiotic-Antimycotic ± TG (2 μM final) were plated in one well of 12-well plate pre-coated with 1 mL of 0.5% bottom agar in the same medium as the top agar. The colonies were grown by changing media with or without TG every 3 days and then visualized by staining with 0.005% crystal violet solution on 15 days after plating or by capturing 10× magnified image using Zeiss Axio Observer Z1. Three biological replicates were performed. In one case, 104 cells were plated, while in the subsequent two experiments, 103 were plated. The stained colonies were counted and the values were normalized to the HCT116 DMSO-treated sample.
3
Anchorage-Independent Growth Assay
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The C666-1 cells were seeded at a density of 5×104 cells/ml in 96-well plates (TPP), followed by cisplatin treatment with or without Nutlin-3. The cells were then plated into a two-layer soft agar made from DNA grade Seakem agarose (Lonza, Rockland, ME, USA) culture system (comprised of a layer of 0.3% agarose in complete media; and with 0.6% agar as a base layer) in 6-well plates (TPP). Anchorage-independent growth was measured by counting the numbers of viable colonies using an Olympus stereomicroscope model SZX7 (Olympus, Tokyo, Japan). The colonies were scored by using Image Pro Plus AMS version 6.3 (Media Cybernetics, Inc. Rockville, MD, USA). Colonies with a minimum diameter of 60 µm, area 2,800 µm2 and roundness score ranging from 0.25 to 0.50 (roundness =4A/πD2; A is the area; D is the maximum diameter; with 1.0 indicating a perfect circle) were counted in order to exclude abortive colonies.
4
Zika Virus Plaque Assay in Vero Cells
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Vero cells were seeded at 3x105 cells/well in a six well plate in DMEM (Gibco) containing 2% FBS (Atlanta Biologicals). Following an overnight incubation at 37°C with 5% CO2, cells were infected with ZIKV-FLR that was serially diluted (10−2 to 10−6) in DMEM with 2% FBS. Virus was removed after one hour, and cells were overlaid with 3ml of a 1:1 mix of 2% Seakem agarose (Lonza) and 2X DMEM with 10% FBS. After three days, cells were overlaid with 3 ml of a 1:1 mix of 2% Seakem agarose and 2x DMEM with 10% FBS and 0.03% neutral red [15 (link)]. Cells were monitored daily for plaque formation, until day 21.
5
Quantification of dsDNA using PicoGreen
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TMOS was purchased from Sigma–Aldrich (St. Louis, MO, USA). TE and PBS buffers were purchased from Teknova (Hollister, CA, USA), and TBE buffer was purchased from Fischer BioReagents (Waltham, MA). Quant-iTTM PicoGreen® dsDNA Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA), and agarose gel was made using SeaKem Agarose (Lonza, Basel, Switzerland). Escherichia coli DNA was purchased from Affymetrix (Santa Clara, California, USA). qPCR was performed on an Applied Biosystems® AB 7500 Fast Dx Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA).
6
PCR Amplification of Recombinant DNA
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PCR of DNA extracted from the clones (adherent or suspension cultures) was performed using GoTaq Flexi DNA polymerase (Promega) to amplify genomic recombination junctions using the primers listed in the figure descriptions and 500 ng of genomic DNA from each recombinant clone or parental cells as a template in 50 μL reactions. The thermal cycling parameters for the PCR was as follows: initial denaturation at 95°C for 2 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min per kb, and a final step of 72°C for 5 min. The PCR products were analysed by electrophoresis in 0.8% agarose (Seakem Agarose, Lonza, USA) gels in 0.5X TBE (Tris-Boric acid-EDTA buffer) containing 0.5 μg/ml ethidium bromide. PCR-generated products were compared with DNA standard markers and digitally documented under UV illumination (Quantum Vilber Lourmat, Germany). PCR-amplified products were analysed by sequencing. Primers are listed in Supplementary Table S1 .
7
Arabidopsis Sulfate Transporter Mutants
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The A. thaliana (L.) Heynh. accession Col-0 was used as a wild type. The mutant seeds sultr1;1 (salk_093256c), sultr1;2 (salk_122974), sultr1;3 (salk_018910), sultr2;1 (salk_109907c), sultr2;2 (salk_111268c), sultr3;1 (salk_127024c), sultr3;2 (salk_023980c), sultr3;3 (salk_000822c), sultr3;4 (salk_100362), sultr3;5 (salk_128559), sultr4;1 (salk_103873c), sultr4;2 (salk_103827c), and cad1-3 (CS68125) were obtained from the ABRC (Arabidopsis Biological Resource Center, https://abrc.osu.edu/ ) and aos (CS6149) and jar1-1 (CS8072) were obtained from the NASC (Nottingham Arabidopsis Stock Centre, http://nasc.nott.ac.uk ). Seeds were surface-sterilized with 70% (v/v) ethanol and 0.05% (v/v) Triton X-100 and sown on media containing 2 mM Ca(NO3)2, 0.5 mM phosphoric acid, 0.75 mM MgSO4, 50 μM H3BO3, 10 μM MnCl2, 2 μM ZnSO4, 1.5 μM CuSO4, 0.075 μM NH4Mo7O24, and 74 μM Fe-EDTA, pH 5.8 with Ca(OH)2, 1% (w/v) sucrose and 1% (w/v) of SeaKem agarose (Lonza) or agarose L03 (TaKaRa) supplemented with designated concentrations of KCl, CsCl and other chemicals. For sulphur supply, CaSO4 was supplemented in addition to 0.75 mM MgSO4 in the base media to add up to the indicated concentrations. After stratification for three to four days at 4°C, plants were placed in a growth cabinet at 22°C in a 16 h light/8 h dark photocycle with a light intensity of 70–90 μmol m−2 s−1.
8
Quantifying Viral Loads in Infected Livers
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Parts of infected livers were weighed, homogenized using ceramic beads, and centrifuged for 10 min at 450 g at 4°C. Supernatant was serially diluted in Dulbecco’s modified Eagles Medium (DMEM, Lonza) supplemented with 10% FCS before inoculation onto overnight grown 90% confluent VeroE6 cells in 6 well plates (Corning). After 1 hour incubation, each 10^2 to 10^7 serial diluted inocula was removed and the cell layers were covered with a solution of 0.5% SeaKem®Agarose (Lonza) in Minimal essential Medium Eagle (EMEM, Lonza) supplemented with 10% FCS. The agarose layer was removed after 5 days incubation at 37°C, 5% CO2, and cells were stained with a crystal violet solution in 2% formaldehyde (Merck). The number of plaques at each dilution was counted and averaged to obtain the viral concentration of the sample in PFU/ml. Each sample was corrected for liver weight input and is expressed as PFU per gram liver.
9
Peptide-Mediated Antiviral Assay
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Lyophilized peptides were resuspended in PBS and stored at 4°C until use. Fibroblasts were seeded into 24-well dishes. After cells reached ∼80% confluence, medium was removed and cells were washed with PBS. Peptide in PBS plus 10% FBS or PBS plus 10% FBS alone as the control was added, and the mixture was incubated for 30 min at 37°C. Virus was added to treated and untreated control wells (∼100 PFU/well) and incubated for an additional hour. Following virus incubation, medium was removed and the overlay was added. Overlays consisted of 0.75% carboxymethyl cellulose (CMC; Sigma-Aldrich, St. Louis, MO) for MCMV-infected wells and 0.5% SeaKem agarose (Lonza, Rockland, ME) in complete medium for HCMV-infected wells. For MCMV assays, plates were incubated for 5 days, at which point they were stained with Coomassie stain and plaques counted using a dissecting microscope. Reduction in viral infectivity was expressed as a percentage of infectivity of PBS-treated wells. Data were analyzed using Prism 7 (GraphPad Software, La Jolla, CA).
10
Molecular Analysis of Spider Prey
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Spiders were identified to species, when possible, and whole‐body DNA extractions were performed using QIAGEN DNeasy Tissue Kits (QIAGEN Inc.) following the manufacturer's animal tissue protocol. The DNA extracted from spiders was then screened for the presence of prey DNA using a general Collembola (hereafter referred to as springtails) primer (Chapman, Schmidt, Welch, & Harwood, 2013 ). PCR procedures, as described by Chapman et al. (2013 ), were followed which optimized the primers and screened for cross‐reactivity against 155 nonspringtail species. Positive tests for springtails in the diet of spiders were determined by electrophoresis of 10 μl of PCR product in 2% SeaKem agarose (Lonza) stained with 0.1 mg/μl GelRed™ (Biotium, Inc.). Even though flies and springtails are most commonly captured by sundews (Ellison & Gotelli, 2009 ), ground‐dwelling spiders primarily consume springtails (Chapman et al., 2013 ; Harwood, Sunderland, & Symondson, 2001 , 2003 ). Because of this, springtails are the most likely common prey for sundews and spiders in this study; thus, molecular analysis for flies in spider gut content was not performed.
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