Supernates collected from one-step virus infections were thawed and irradiated with 7000 J/m2 of UVC irradiation over 20 minutes to inactivate infectious virus. The supernates were then assayed for paracrine antiviral signaling molecules as previously described (Voigt et al., 2013 (link)). Briefly, serial 1:2 dilutions were incubated over A549 cells in 96-well plates for 24 hours, and the antiviral state of the cells was then challenged by infection with VSV-DsRed2 virus at MOI 5. After 24 hours incubation, successful infection as indicated by DsRed2 fluorescence was read using a GE Typhoon FLA 9000 Biomolecular Imager at 555/580 nm under BSL2 conditions. Inhibition of virus replication indicated presence of antiviral signaling molecules, the concentration of which was determined by calculating the IC50 dilution and presented in units (U) per ml in the original one-step virus infection. Here, one unit of antiviral activity per ml is the amount that inhibits 50% of VSV replication in A549 cells using the assay described above.
Typhoon fla 9000 biomolecular imager
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The Typhoon FLA 9000 is a biomolecular imager designed for high-performance fluorescence and chemiluminescence detection. It provides sensitive and quantitative imaging of a variety of samples, including gels, blots, and microarrays.
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Lab products found in correlation
Neg772002mc, by PerkinElmer (2 mentions)
Storage phosphor screen, by GE Healthcare (2 mentions)
Hybond, by Cytiva (1 mentions)
Adenylate 32p nad, by PerkinElmer (1 mentions)
Whatman, by Cytiva (1 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
Model 583, by Bio-Rad (1 mentions)
Qubit rna iq assay kit, by Thermo Fisher Scientific (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Zeta probe blotting membrane, by Bio-Rad (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Imagequant tl 8, by GE Healthcare (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Amersham typhoon biomolecular imager, by Cytiva (1 mentions)
Amersham typhoon, by Cytiva (1 mentions)
13 protocols using typhoon fla 9000 biomolecular imager
1
Quantifying Antiviral Paracrine Signaling
2
Autophagy Induction and Inhibition Assay
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OECM1 and SAS cells were plated into 96-well plates at a cell density of 5 × 103/well. The cells were incubated at 37 °C and 5% CO2. OECM1 and SAS cells were incubated in 0.4 μM of PG and 0.4 μM of PG plus 5 mM of 3-methyladenine (3MA) for 24 h. Then, cells were fixed with 3.7% formaldehyde/PBS and stained by the Alexa Fluor 488-conjugated anti-LC3-II rabbit antibody (Thermo-Fisher). Images were captured by a Typhoon™ FLA 9000 Biomolecular Imager (GE Healthcare, Little Chalfont, UK). The fluorescence focus units were quantified in each well.
3
Northern Blotting of Yeast tRNA
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Northern blotting was performed as earlier reported (11 (link)). Briefly, the BY4741 Δdtd strains expressing EcDTD (WT), EcDTD-Mut (A102F), empty vector, and ScDTD were grown in 5 ml of -Leu synthetic defined broth medium as primary culture and later inoculated in 20 ml of secondary culture with 10 μM Cu2+ for 24 hours at 30°C. The culture was pelleted and total RNA was isolated under acidic conditions in ice (41 (link)).
This extracted RNA (0.15 to 0.25 A260 unit) was run on 6.5% acid–urea PAGE at 4°C for 20 to 24 hours. The RNA from the gel was electroblotted on Hybond-N+ nitrocellulose (GE Healthcare) membrane at 15 V, 3 A for 38 min. This membrane with nucleic acids transferred on was ultraviolet cross-linked for 4 min. This was followed by hybridization with [32P]-labeled tRNA-specific DNA oligo probe. The probe was labeled using [Ƴ-32P]ATP using polynucleotide kinase enzyme (NEB). The signal from the probe was recorded overnight on an image plate and quantified using a Typhoon FLA 9000 biomolecular imager (GE Healthcare).
This extracted RNA (0.15 to 0.25 A260 unit) was run on 6.5% acid–urea PAGE at 4°C for 20 to 24 hours. The RNA from the gel was electroblotted on Hybond-N+ nitrocellulose (GE Healthcare) membrane at 15 V, 3 A for 38 min. This membrane with nucleic acids transferred on was ultraviolet cross-linked for 4 min. This was followed by hybridization with [32P]-labeled tRNA-specific DNA oligo probe. The probe was labeled using [Ƴ-32P]ATP using polynucleotide kinase enzyme (NEB). The signal from the probe was recorded overnight on an image plate and quantified using a Typhoon FLA 9000 biomolecular imager (GE Healthcare).
4
Quantification of DENV-2 Inhibition by Honokiol
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The BHK and Huh7 cells were first infected with DENV-2 by incubation with medium containing the virus for 1 h. After infection, the medium was replaced with virus-free medium containing a different concentration of honokiol and incubated for 48 h. The supernatant were collected, diluted 10-fold serially with serum free medium, and added to a 96-well plate with confluent BHK cells. After 1 h of incubation, 100 µL of DMEM/FBS containing 0.8% methyl cellulose was added to each well and the cells were cultured at 37°C with 5% CO2 for 72 h. The resulting cells were washed with PBS, fixed with 3.7% formaldehyde/PBS, and then permeabilized with 0.1% Triton X-100/PBS. The anti-DENV-2 NS1 antibody 206-35 was added and incubated for 1.5 h. The cells were then washed and incubated with secondary HRP-conjugated goat anti-mouse IgG antibody (1:1000, GeneTex) for 1.5 h at room temperature. After extensive washes, tyramide-fluorescein [61 (link)] was added to each well and the fluorescent foci were analyzed by the Typhoon FLA 9000 Biomolecular Imager (GE Healthcare, Pittsburgh, PA, USA).
5
ADP-ribosylation and Hydrolysis Assays
6
Optimization of PCFT Expression in Sf9 Cells
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For optimization of PCFT expression, Sf9 cells in suspension were grown at 27°C in 250-ml baffled flasks containing 50 ml of HyClone CCM3 medium (GE Healthcare Life Sciences, Pittsburgh, PA), shaken at 125 rpm. The cells were infected at a density of 2 x 106 cells/ml using a multiplicity of infection (MOI) of 2. One-ml samples were collected at indicated times to measure viability (Trypan blue staining), and PCFT expression was evaluated by Western blot using an antibody against the His6 tag at the C-terminus of the recombinant PCFT. After incubation with goat anti-mouse secondary antibody, Alexa Fluor 680 (Thermo Fisher Scientific), the signal was visualized using an imager (Odyssey Infrared Imager Li-Cor Biosciences, Lincoln, NE; or Typhoon FLA 9000 Biomolecular Imager, GE Healthcare Life Sciences). For PCFT expression, Sf9 cells were grown at 27°C in 2-l baffled flasks containing 750 ml of HyClone CCM3 medium. Cells were harvested by centrifugation (1,000 g for 15 min) ~48 h post-infection, when cell viability determined by Trypan blue staining was 40%.
7
Pulse Labeling of Newly Translated Proteins
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For the purpose of pulse labeling of newly translated proteins, 50 isolated islets were incubated in complete RPMI media without cysteine and methionine (MP Biomedicals, #SKU 091646454) for 1 h. Subsequently media was supplemented with 250 μCi of [35S]-cysteine/methionine mixture (PerkinElmer, NEG772002MC) and islets were incubated under normal conditions for 30 min. Islets were then lysed and proteins separated by SDS-gel electrophoresis as described above. Gels were fixed for 30 min in 50% (v/v) ethanol in water with 10% (v/v) acetic acid, dried in gel dryer (Bio-Rad model 583) and then exposed to storage phosphor screen (GE Healthcare) overnight. Screens were imaged and digitized using Typhoon FLA 9000 biomolecular imager (GE Healthcare). Protein bands intensity was quantified with Adobe Photoshop software version 7.
8
Antivirals Induced by Virus Strains
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Separate wells of LNCaP and PC-3 cells were infected with each ancestor and evolved virus strain, in triplicate, and the infection was allowed to progress for 24 hpi. Supernatants containing secreted paracrine-signaling antivirals were collected and frozen for later assay. Secreted antivirals induced in either LNCaP or PC-3 cells by the ancestor and evolved viral strains were titered for antiviral paracrine-signaling activity. Upon thawing, active virus in the supernate samples was inactivated by exposure to 7000 J/m2 UVC irradiation with rocking over 20 min. Samples were then assayed for antiviral activity using published methods (Voigt et al., 2013 (link)). Briefly, samples were diluted serially 1:2, incubated for 24 h over A549 human lung epithelial cells (American Type Culture Collection strain #CCL-185) responsive to human antivirals, and challenged with a DsRed2-VSV reporter virus. After 24 h incubation, assay plates were scanned using a GE Typhoon FLA 9000 Biomolecular Imager at 555/580 nm. Mean fluorescent intensity of assay wells, in duplicate, was measured using JEXTools and used to calculate viral inhibitory activity present in the assayed antiviral secretions.
9
Quantifying RNA Transcript Levels
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The levels of transcripts were assessed by Northern blot analysis. Overnight cultures were diluted 100-fold in 10 mL of fresh medium and grown at 26 °C for about 6 h (until OD600 = 1). Total RNA was isolated using TRItidy™ (AppliChem, Darmstadt, Germany), as described previously [109 (link)]. The quality of the RNA was checked by gel electrophoresis and quantified using a Qubit 4 Fluorometer with a Qubit RNA IQ Assay Kit (Thermo Fisher Scientific). For each sample, 10 µg of total RNA was separated by denaturing polyacrylamide gel electrophoresis (6% acrylamide/bis 29:1, 7 M urea). The RNA was then electro-transferred to a Zeta-Probe® Blotting Membrane (Bio-Rad, Hercules, CA, USA). Crosslinking was performed by UV irradiation (1200 J, UVP HL-2000 HybriLinkerTM, Analytik Jena, Jena, Germany). The membrane was then incubated with DNA oligonucleotide probes labeled with 32P (Table S2 ). After washing to remove unbound probes, membranes were exposed to phosphor storage screens and analyzed using a Typhoon FLA 9000 biomolecular imager (GE Healthcare). Quantification was performed using ImageQuantTL (version 8.2, GE Healthcare). The presented blots are representative of at least two independent experiments.
10
Northern Blot Analysis of 11-kDa mRNA
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Northern blot analysis was performed as previously described (72 (link), 73 (link)). In brief, total RNA was extracted from pM20-transfected or pM20ISE3-mut7-transfected UT7/Epo-S1 cells by the use of TRIzol reagent (Invitrogen). A 5-μg volume of the total RNA was separated on a denatured 1.4% agarose gel and visualized using ethidium bromide (EB) staining. The stained 28S ribosome RNA band served as a loading control. For the detection of 11-kDa mRNA, the blot was hybridized with a [32P]dCTP-labeled DNA cap probe (nt 4619 to 5147), which was amplified from pM20 by PCR. Hybridization signals were captured by the use of a storage phosphor screen and visualized on a Typhoon FLA 9000 biomolecular imager (GE Healthcare). Quantification of the bands was carried out using ImageQuant TL 8.1 software (GE Healthcare).
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