Recombinant adenovirus expressing the βgal protein under the control of the human CMV promoter (Ad-LacZ) and lacking E1 and E3 genes was used (19). Ad-LacZ was propagated on permissive HER-911 cells and was purified with the Vivapure AdenoPack 20 (Sartorius, Stedim biotech, 13781 Aubagne Cedex, France) according to the manufacturer's specifications. Virus titer was determined in a cytopathic effect assay. Ad-LacZ was stored at −80°C in PBS and injected intravenously at a dose of 2×109 pfu.
Vivapure adenopack 20
Manufactured by Sartorius
Sourced in Germany
The Vivapure AdenoPack 20 is a lab equipment product offered by Sartorius. It is a device designed for the purification of adenoviral vectors. The core function of this product is to facilitate the concentration and purification of adenoviral samples.
Automatically generated - may contain errors
8 protocols using vivapure adenopack 20
1
Adenoviral Vector for in vivo Gene Delivery
2
Adenoviral shRNA Knockdown of Nmnat2 in Mouse Cells
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Adenoviral shRNA expression constructs targeting mouse Nmnat2 mRNA and scramble shRNA were purchased from VectorBuilder. Adenoviruses were generated by transfection of the plasmid constructs into AD293 cells (Agilent) using Lipofectamine 2000 (Thermo). The viruses were collected according to the manufacturer's protocol and purified by using Vivapure AdenoPACK 20 (Sartorius).
For the measurement of insulin secretion and content, the cells were plated in 24-well plates at 2 × 105 cells/well. For the other experiments, the cells were plated in 6-well plates at 4 × 105 cells/well. Following a 4-day culture, the cells were infected with adenoviruses at multiplicity of infection (MOI) of 5. Forty-eight hours after the first infection, the culture medium was discarded and the cells were again infected with the same amount of viruses. Forty-eight hours after the second infection, the cells were subjected to analysis.
For the measurement of insulin secretion and content, the cells were plated in 24-well plates at 2 × 105 cells/well. For the other experiments, the cells were plated in 6-well plates at 4 × 105 cells/well. Following a 4-day culture, the cells were infected with adenoviruses at multiplicity of infection (MOI) of 5. Forty-eight hours after the first infection, the culture medium was discarded and the cells were again infected with the same amount of viruses. Forty-eight hours after the second infection, the cells were subjected to analysis.
3
Adenoviral Vector Construction for RSV-F Protein
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The vaccine vector encoding RSV-F was constructed by insertion of the codon-optimized full-length sequence of the RSV-A2 F protein (GenBank database entry EF566942) as described before (33 (link)). The murine mature IL-1β-encoding vector and the Ad-empty control were constructed as described elsewhere (16 (link)). For the generation of Ad-IPS-1, the full-length cDNA sequence (NCBI Ref Seq NM_001206385.1) was initially cloned into the pShuttle vector. Replication-deficient adenoviral vectors were produced according to the AdEasy adenoviral vector system (34 (link)). Viral particles were purified and concentrated with the Vivapure Adenopack20 (Sartorius). The concentration of total Ad was measured by optical density at 260 nm (OD260) and infectious particles by Reed-and-Muench’s tissue-culture infectious dose 50 assay (35 ). Ratios of total to infectious particles usually were in the range of 200:1.
4
Viral and Bacterial Infection Models
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MCMV strain (Strain Smith; ATCC: VR194) was used and kindly provided by Professor U.H. Koszinoswki, Department of Virology, Max von Pettenkofer Institute. MCMV was propagated and titrated on NIH 3T3 cells (ECACC), stored at -80°C, and injected i.v. at a dose of 2x106 pfu.
Recombinant adenovirus expressed the β-gal protein under the control of the human CMV promoter (Ad-lacZ [63 (link)]). Ad-LacZ was propagated on HER-911 cells and purified with the Vivapure AdenoPack 20 (Sartorius; Stedim Biotech). Virus titer was determined in a cytopathic effect assay [63 (link)]. Ad-LacZ was stored at -80°C in PBS and injected i.v. at a dose of 2x109pfus. Vaccinia virus (VVWR, ATCC: VR1354) was injected i.v. at 2x106 pfu. Listeria-OVA was injected i.v. at a dose of 1x103cfu.
Recombinant adenovirus expressed the β-gal protein under the control of the human CMV promoter (Ad-lacZ [63 (link)]). Ad-LacZ was propagated on HER-911 cells and purified with the Vivapure AdenoPack 20 (Sartorius; Stedim Biotech). Virus titer was determined in a cytopathic effect assay [63 (link)]. Ad-LacZ was stored at -80°C in PBS and injected i.v. at a dose of 2x109pfus. Vaccinia virus (VVWR, ATCC: VR1354) was injected i.v. at 2x106 pfu. Listeria-OVA was injected i.v. at a dose of 1x103cfu.
5
Adenoviral Vector Purification and Concentration
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ADV was purified by using the Vivapure AdenoPACK 20 purification kit (Sartorius AG, Weender Landstr, Göettingen, Germany) [6 (link)]. The ADVs were concentrated by ultrafiltration to yield 2 × 1011–2 × 1012 viral particles per mL (VP/mL) in PBS (-). ADVs were stocked at –80°C for up to 2 months.
6
Adenovirus-Mediated Ski Overexpression in Fibroblasts
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The Ski‐overexpressing recombinant adenovirus Adeno‐MCMV‐SKI‐3Flag‐P2A‐EGFP (Ad‐Ski) and the empty control recombinant adenovirus Adeno‐MCMV‐3Flag‐P2A‐EGFP (Ad‐EGFP) were purchased from Obio Technology Corp., Ltd. Human Ski cDNA (GenBank accession number NM_003036.3) was packaged into the adenovirus vector. The virus was amplified in human embryo kidney 293 (HEK293) cells, purified by Vivapure Adeno PACK 20 (Sartorius), and titrated as previously described.15 The efficiency of Ad‐Ski infection in fibroblasts was demonstrated by Western blotting and immunofluorescence. Primary fibroblasts were treated with HG and transiently transfected with Ad‐EGFP or Ad‐Ski for 48 hour, following the manufacturer's instructions.
7
MCMV and Recombinant Adenovirus Infection Protocol
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MCMV (Strain Smith, ATCC: VR194) was kindly provided by Professor U.H. Koszinowski, Department of Virology, Max von Pettenkofer Institute, Germany. MCMV was propagated and titrated on NIH 3T3 cells (ECACC), stored at -80°C and injected i.v. at a dose of 1 x 10
6 pfus. Recombinant adenovirus expressed the βgal protein under the control of the human CMV promoter (AdLacZ
8 (link); kindly provided by Dr. S. Rusconi, University of Fribourg, Fribourg, Switzerland) was propagated on HER-911 cells and purified with the Vivapure AdenoPack 20 (Sartorius, Goettingen, Germany, catalog no 14-558-548). Virus titer was determined in a cytopathic effect assay. In brief, serial dilutions of the adenovirus were used to infect HER-911 cells on a 96-well microtiter plate, and cytopathic effect was determined after 5 d by microscopy. Tissue culture infectious dose of 50% was calculated by the Reed–Muench method. AdLacZ was stored at -80°C and injected i.v. at a dose of 2x10
9 pfus.
6 pfus. Recombinant adenovirus expressed the βgal protein under the control of the human CMV promoter (AdLacZ
8 (link); kindly provided by Dr. S. Rusconi, University of Fribourg, Fribourg, Switzerland) was propagated on HER-911 cells and purified with the Vivapure AdenoPack 20 (Sartorius, Goettingen, Germany, catalog no 14-558-548). Virus titer was determined in a cytopathic effect assay. In brief, serial dilutions of the adenovirus were used to infect HER-911 cells on a 96-well microtiter plate, and cytopathic effect was determined after 5 d by microscopy. Tissue culture infectious dose of 50% was calculated by the Reed–Muench method. AdLacZ was stored at -80°C and injected i.v. at a dose of 2x10
9 pfus.
8
Adenoviral Ski Overexpression in FLSs
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The Ski-overexpressing recombinant adenovirus Adeno-MCMVSKI-3Flag-P2A-EGFP (Ad-ski) and the empty control recombinant adenovirus Adeno-MCMV-3Flag-P2A-EGFP (Ad-EGFP) were purchased from Obio Technology Corp., Ltd. Human Ski cDNA (GenBank accession number NM_003036.3 ) was packaged into the adenovirus vector. The virus was amplified in human embryo kidney 293 (HEK293) cells, purified by Vivapure Adeno PACK 20 (Sartorius), and titrated as previously described [19 (link)]. The efficiency of Ad-Ski infection in FLSs was demonstrated by Western blotting and immunofluorescence. FLSs were transiently transfected with Ad-EGFP or Ad-ski for 48 h, following the manufacturer's instructions. Ski-overexpressing recombinant adenovirus of Ski was obtained from Professor Ping Li of the Chinese Army Characteristic Medical Center.
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