Expression plasmids for pCAGGS-EBOV (Mayinga), SUDV (Boniface), BDBV (Butalya), or RESTV (Pennsylvania 89) sGP were transfected in 293F cells according to the manufacturer’s instructions. After incubation for 4 days, the supernatant was cleared from cell debris by centrifugation twice at 3500 rpm for 10 min at 4 °C. The supernatants containing each sGP were collected and concentrated in Amicon Ultra 30 K filters (Millipore) to a final concentration of 1–2 mg/mL and stored at −80 °C. Supernatant of nontransfected 293F cells was also concentrated using the same method and used as a control protein. Expression of each sGP was confirmed by mixing the concentrated protein 1:1 with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 20% β-mercaptoethanol and incubated at 99 °C for 10 min. SDS-PAGE with all samples was performed in parallel on Tris-Glycine eXtended (TGX) criterion pre-cast gels (Bio-Rad Laboratories). The gel was processed for silver staining, using the Pierce Silver Stain Kit (Thermo Fisher Scientific, USA).
Amicon ultra 30 k filters
Manufactured by Merck Group
The Amicon Ultra 30 K filters are a type of lab equipment used for the ultrafiltration and concentration of macromolecules, such as proteins, antibodies, and nucleic acids. The filters have a molecular weight cut-off (MWCO) of 30,000 Daltons, which means they can effectively retain molecules larger than 30 kDa while allowing smaller molecules to pass through. This allows for the concentration and purification of the desired macromolecules from a sample.
Automatically generated - may contain errors
Lab products found in correlation
Aav helper free system, by Agilent Technologies (3 mentions)
Hek293 cell, by American Type Culture Collection (2 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Icap 6300 duo view spectrometer, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Surescan microarray scanner, by Agilent Technologies (1 mentions)
6 protocols using amicon ultra 30 k filters
1
Recombinant Ebolavirus sGP Production
2
AAV Vector Construction and Delivery
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Vector construction, production, and in vivo delivery of adeno-associated virus (AAV) were performed based on the AAV helper-free system (Agilent, San Francisco, Calif). The recombinant adenoviral vector, pAAV-ITR-HEF1, pAAV-ITR-AurKA, pAAV-ITR-Cep97, pAAV-ITR-CP110, pAAV-ITR-shVHL, pAAV-ITR-shGSK3β, and pAAV-ITR-shIFT88 were constructed by cloning the cDNA encoding region or shRNA sequences into pAAV-ITR. The vector pAAVITR-GFP encoding green fluorescence protein was used as a negative control. Recombinant AAVs were produced by HEK293 cells (ATCC) transfected with pAAV-ITR vectors together with pAAV-RC and pHelper plasmids, and then purified by discontinuous iodixanol gradient centrifugation. Purified recombinant AAVs were concentrated and desalted by centrifugation through Amicon Ultra 30K filters (Millipore, Billerica, Mass). For in vivo delivery, recombinant AAVs equivalent to 1.0 × 1012 viral genome copy were delivered though mouse tail vein.
3
AAV Vector Construction and Delivery
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Vector construction, production and in vivo delivery of adeno-associated virus (AAV) were performed based on the AAV helper-free system (Agilent). The recombinant adenoviral vector pAAV-IL38 was constructed by cloning the cDNA encoding region into pAAV-ITR. The vector pAAV-GFP encoding green fluorescence protein was used as a negative control. Recombinant AAVs were produced by HEK293 cells (ATCC) transfected with pAAV-ITR vectors together with pAAV-RC and pHelper plasmids, and then purified by discontinuous iodixanol gradient centrifugation. Purified recombinant AAVs were concentrated and desalted by centrifugation through Amicon Ultra 30 K filters (Millipore). For in vivo delivery, recombinant AAVs equivalent to 1.0 × 1012 viral genome copies were delivered via the mouse tail vein.
4
AAV-Mediated In Vivo Gene Delivery
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Vector construction, production, and in vivo delivery of adeno-associated virus (AAV) were performed based on the AAV helper-free system (Agilent). The recombinant adenoviral vector pAAV-IL35 (EBI3-2A-IL-12p35) was constructed by cloning the cDNA encoding region into pAAV-ITR. The vector pAAV-GFP encoding green fluorescence protein was used as a negative control. Recombinant AAVs were produced by HEK293 cells (ATCC) transfected with pAAV-ITR vectors together with pAAV-RC and pHelper plasmids, and then purified by discontinuous iodixanol gradient centrifugation. Purified recombinant AAVs were concentrated and desalted by centrifugation through Amicon Ultra 30K filters (Millipore). For in vivo delivery, recombinant AAVs equivalent to 1.0 × 1012 viral genome copies were delivered though mouse tail vein.
5
Quantifying DNA-Cation Interactions
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Buffer-equilibration
and ion counting were carried out as reported previously.48 (link),49 (link) Briefly, DNA was purified as described above, followed by hybridization
in 160 mM K+ at the pH of the experiment. Successive buffer
exchanges (eight rounds) with the appropriate ionic conditions and
pH were carried out at 4 °C using Amicon Ultra-30K filters (Millipore,
MA). Aliquots of the DNA-containing sample, the flow-through, and
the buffer were diluted into 5 mL of 50 mM ammonium acetate (pH 5.0).
Dilution factors were determined so that the phosphorus (from DNA),
potassium, and magnesium concentrations were within the linear range
of ICP-OES detection. The concentrations in each solution were determined
simultaneously using an ICAP 6300 Duo View Spectrometer (Thermo Scientific,
USA) calibrated with ICP standards (Spex Certiprep, USA). The number
of cations associated with a DNA was calculated usingeq 3 , where Cion is
the cation concentration of the DNA-sample and buffer, respectively,
and CDNA is the DNA concentration. The
number of anions excluded by the DNA was not quantified because Cl– concentration is not measurable in this experimental
system.
and ion counting were carried out as reported previously.48 (link),49 (link) Briefly, DNA was purified as described above, followed by hybridization
in 160 mM K+ at the pH of the experiment. Successive buffer
exchanges (eight rounds) with the appropriate ionic conditions and
pH were carried out at 4 °C using Amicon Ultra-30K filters (Millipore,
MA). Aliquots of the DNA-containing sample, the flow-through, and
the buffer were diluted into 5 mL of 50 mM ammonium acetate (pH 5.0).
Dilution factors were determined so that the phosphorus (from DNA),
potassium, and magnesium concentrations were within the linear range
of ICP-OES detection. The concentrations in each solution were determined
simultaneously using an ICAP 6300 Duo View Spectrometer (Thermo Scientific,
USA) calibrated with ICP standards (Spex Certiprep, USA). The number
of cations associated with a DNA was calculated using
the cation concentration of the DNA-sample and buffer, respectively,
and CDNA is the DNA concentration. The
number of anions excluded by the DNA was not quantified because Cl– concentration is not measurable in this experimental
system.
6
Comparative Genomic Hybridization of Chondrocytes
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Comparative genomic hybridization (CGH) array analysis was performed on chondrocytes at different passages (Supplementary Table S3 ). DNA was extracted using the QIAGEN QIAamp DNA Mini Kit (Qiagen, Hilden) according to the manufacturer’s protocol. Array CGH was performed using the Agilent SurePrint G3 Human CGH Microarray kit 4_44K (design ID 014950) with 43 kb overall median probe spacing (Agilent Technologies). Practical resolution was approximately 200 kb. Labeling and hybridization were performed following the protocols provided by the manufacturers. Briefly, sample DNA and DNA of a sex-matched control (1 µg of each) was labeled with Cy3-dUTP and Cy5-dUTP, respectively (Sure Tag Labelling Kit, Agilent Technologies). Labeled products were purified by Amicon Ultra 30 K filters (Millipore). Hybridization was performed according to the protocol provided by Agilent. Sample and control DNA was pooled and hybridized with 2 mg of Human Cot-I DNA at 65 °C with rotation for 24 h. Arrays were analyzed using an Agilent SureScan Microarray scanner and the Agilent Feat. A graphical overview was obtained using Cytogenomics 5.1.2.1 software and used to determine genetic stability (absence of deletions and duplications). Data analysis was done on UCSC Genome Browser Human Genome build19.
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