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Related assays
18 Assays visible to you, out of a total of 24ZIKV RNA was extracted from virus working stocks using the QIAmp viral RNA minikit (Qiagen, Hilden, Germany). Isolated RNA was then digested with DNase I and purified using the Qiagen RNeasy kit. RNA quality was analyzed on the 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) using the Agilent RNA 6000 Pico assay and quantified on a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) prior to rRNA depletion and RNA library prep using the KAPA HyperPrep kit (Roche Diagnostics Corporation, Indianapolis, ...
Submitter: John Cornelius
Assay type: RNA Sequencing
Technology type: Rna-seq
Investigation: Zika Virus
Hybridoma supernatant was harvested as the source of ZV-13 antibody once ~95% cell death was observed. The supernatant was clarified by centrifuging for 10 min at 400 × g at 4°C and then filtered with a 0.22-μm filter prior to antibody purification via protein A affinity chromatography using the Pierce Protein A IgG purification kit and NAb Protein A Plus spin columns (all from Thermo Fisher-Life Technologies). Purified ZV-13 antibody was concentrated and buffer-exchanged into 1× phosphate-buffered ...
Submitter: John Cornelius
Assay type: Immunoassay
Technology type: Affinity Chromatography
Investigation: Zika Virus
A549 cells were seeded in 96-well plates at a density of 2 × 104 cells per well. The following day, virus stocks were serially diluted in DMEM containing 2% FBS (2% DMEM), and cells were inoculated with dilutions in triplicate at 37°C for 2 h. After the incubation, cells were overlaid with DMEM supplemented with 1% carboxymethylcellulose (Fisher Scientific), 2% FBS, 10 mM HEPES, and 1× penicillin/streptomycin. At 48 hpi, the methylcellulose overlay and medium were removed, and cells were fixed ...
Submitter: John Cornelius
Assay type: Organism or Strain Characteristics
Technology type: Immunostaining
Investigation: Zika Virus
A549 cells were seeded in 6-well plates at a density of 6 × 105 cells per well. The following day, virus stocks were serially diluted in 2% DMEM, and cells were inoculated and fixed as described above. Following fixation, cells were washed with 1× PBS, and an anti-flavivirus envelope antibody, 4G2 (Ab00230-2.0; Absolute Antibody, Oxford, UK), diluted 1:500 in permeabilization/wash/block buffer (see recipe above) was added to the cells. Following incubation with primary antibody, donkey anti-mouse ...
Submitter: John Cornelius
Assay type: Immunoassay
Technology type: Imaging
Investigation: Zika Virus
A549 cells were seeded at a density of 3 × 105 cells per well in 12-well plates. Cells were uninfected, mock-infected, or infected with ZIKV variants the next day at an MOI of 5 FFU/cell, and virus inocula prepared in DMEM were allowed to adsorb at 37°C for 2 h with rocking. Uninfected samples are seeded cells that remained untouched throughout the infection time course, whereas mock-infected samples were inoculated with DMEM. Following adsorption, the inocula were removed, and cells were washed ...
Submitter: John Cornelius
Assay type: Cultivation Experiment
Technology type: Technology Type
Investigation: Zika Virus
A549 cells were seeded at a density of 3 × 105 cells per well in 12-well plates. Cells were precooled at 4°C prior to the addition of ice-cold virus inoculum. Inocula were allowed to adsorb to cells at 4°C for 1 h. Inocula were then removed, and cells were washed with ice-cold 1× PBS before fresh prewarmed cDMEM was added to cells. Cells were returned to 37°C until the appropriate time points to evaluate viral entry, genome replication, and virion production. For viral attachment assays, intracellular ...
Submitter: John Cornelius
Assay type: Cultivation Experiment
Technology type: Technology Type
Investigation: Zika Virus
A549 cells were infected as described above. At the appropriate time points, 1 mL of cell culture supernatants was harvested and centrifuged at 2,000 rpm for 10 min at 4°C. Extracellular RNA was extracted from the clarified supernatants using the QIAmp viral RNA minikit to quantify extracellular viral copy numbers. Cells were washed with ice-cold stringent wash buffer (1 M NaCl, 50 mM sodium bicarbonate, pH 9.5) for 3 min to remove cell surface-associated viruses. After ice-cold 1× PBS washes, ...
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: Technology Type
Investigation: Zika Virus
A549 cells were infected as described above. At the appropriate time points, 1 mL of cell culture supernatants was harvested and centrifuged at 2,000 rpm for 10 min at 4°C. The clarified supernatants were used for plaque assays to quantify extracellular virions. Cells were washed with ice-cold stringent wash buffer as described above and then detached with 0.05% trypsin-EDTA (Fisher Scientific). Cells were resuspended in 1 mL cDMEM and centrifuged at 300 × g for 5 min at 4°C. Cell pellets were ...
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: Technology Type
Investigation: Zika Virus
Vero cells were seeded at a density of 1.85 × 105 cells per well in 6-well plates. The next day, supernatants from infected samples were serially diluted in DMEM containing 1% FBS. Vero cells were incubated with dilutions in duplicate at 37°C for 1 h with rocking. Following incubation, cells were overlaid with 1% low-melt agarose (Fisher Scientific), 5% sodium bicarbonate (Fisher Scientific), and 5% FBS in 2× DMEM (Fisher Scientific). Plaques were visualized 4 to 5 days later with a 1% low-melt ...
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: Technology Type
Investigation: Zika Virus
cDNA was synthesized from isolated RNA using the iScript Select cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). cDNA was diluted to a final concentration of 2.5 ng/μL for TaqMan qRT-PCR on the QuantStudio 5 real-time PCR system (Thermo Fisher) using TaqMan universal PCR master mix (Fisher Scientific) and ZIKV NS3-specific primers and probe (Table 4; IDT, Coralville, IA). The ZIKV NS3-specific probe has a 5′ 6-carboxyfluorescein (FAM) reporter dye and a 3′ Iowa Black FQ quencher. ZIKV NS3-specific ...
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: qRT-PCR
Investigation: Zika Virus
cDNA was synthesized as described above. For each sample, cDNA was diluted to a final concentration of 2.5 ng/μL for SYBR green qRT-PCR on the QuantStudio 5 real-time PCR system using the SYBR green master mix (Thermo Fisher) and gene-specific primers (Table 4; Qiagen; IDT). Each sample was run in triplicate.
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: qRT-PCR
Investigation: Zika Virus
RNA samples were diluted to a final concentration of 20 ng/μL and hybridized to reporters and capture probes from a custom-generated innate immunity probe set (NanoString, Seattle, WA, USA). Following hybridization for 16 h at 65°C, sample preparation was completed on an nCounter MAX prep station (NanoString) according to the manufacturer’s protocol. RNA transcript counts were quantified on a NanoString nCounter (NanoString) according to the manufacturer’s instructions.
Submitter: John Cornelius
Assay type: Amplification
Technology type: Sequencing
Investigation: Zika Virus
Raw reads were assessed for technical quality control flags using the nSolver software (NanoString) and transferred into RStudio (Boston, MA, USA). As the design of our probe set was nonrandom, we utilized a custom approach for data analysis as previously described to account for large differences in the number of observed counts between mock-infected and infected samples (114). Briefly, we used count data within each sample vector as an internal reference, against which we compared genes of ...
Submitter: John Cornelius
Assay type: RNA-seq Profiling
Technology type: Rna-seq
Investigation: Zika Virus
Cells were washed with 1× PBS and lysed on ice with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate) containing freshly added phosphatase inhibitors (1:100; VWR), protease inhibitors (1:100; Sigma-Aldrich), and 250 nM okadaic acid (1:1,000; Fisher Scientific). Cell lysates were collected and stored at −80°C. Lysates were subsequently thawed on ice and sonicated in a water bath containing ice slurry for three 30-s bursts on ...
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: Technology Type
Investigation: Zika Virus
For immunoblot analysis, protein lysates were incubated with 4× Laemmli buffer (Bio-Rad) containing 10% β-mercaptoethanol (Thermo Fisher-Life Technologies) at 95°C for 3 min. Approximately 7 to 10 μg of total protein was loaded per lane onto 4 to 20% Criterion TGX gradient gels (Bio-Rad) and electrophoresed at ~96 V in 1× sodium dodecyl sulfate (SDS) running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Proteins were then transferred onto nitrocellulose membranes (Fisher Scientific) at 90 V for ...
Submitter: John Cornelius
Assay type: Immunoassay
Technology type: Western Blot
Investigation: Zika Virus
A549 cells were seeded on no. 1 glass coverslips in a 24-well plate at a density of 1 × 105 cells per well. The next day, cells were mock infected or infected with ZIKV strains and fixed with 4% formaldehyde (Fisher Scientific) in filtered 1× TBS for 30 min at room temperature. Cells were then washed with filtered 1× TBS and permeabilized and blocked in 5% NGS in 1× TBS (5% NGS/TBS) containing 0.1% Triton X-100 for 30 min at 4°C. For pSTAT1 staining, cells were permeabilized with 100% ice-cold ...
Submitter: John Cornelius
Assay type: Immunoassay
Technology type: Microscopy
Investigation: Zika Virus
Cells were seeded, infected, or transfected and treated with IFN-β as described above. Following IFN-β treatment, A549 RIG-I KO and HEK293T cells were pelleted and fixed with 4% formaldehyde in 1× PBS at room temperature for 15 min. Cells were then washed with excess 1× PBS, pelleted, and resuspended in 1× PBS for storage at 4°C. Fixed cells were permeabilized with 90% ice-cold methanol in 1× PBS for at least 10 min on ice. Cells were washed with excess 1× PBS and incubated for 1 h at room ...
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: Flow Cytometry
Investigation: Zika Virus
Submitter: John Cornelius
Assay type: Experimental Assay Type
Technology type: Technology Type
Investigation: Zika Virus