Sodium salts of FFA’s were purchased from Sigma (St. Louis, MO, USA). SCD1 (A939572) and Ire1α (4μ8c) inhibitors were from Calbiochem (EMD Millipore, Burlington, MA, USA). The processed murine SREBP1c coding sequence was amplified from liver cDNA, sub-cloned into pAdTrack-CMV, sequence verified, and incorporated into pAdEasy-1 vector via homologous recombination in BJ5183 cells (Agilent Technologies, Santa Clara, CA, USA). Total cellular lipids were extracted according to the Folch method [27 (link)] and resolved on a silica gel plate using hexane-diethyl ether-acetic acid (90:30:1) as the developing solvent. Mouse plasma PYY was measured by EIA (RayBiotech, Norcross, GA, USA) and human PYY was measured using RIA as previously described [19 (link)]. Plasma TGs were measured using the L-type triglyceride M kit (Wako Diagnostics).
Padtrack cmv
Manufactured by Agilent Technologies
Sourced in United States
PAdTrack-CMV is a software tool developed by Agilent Technologies for the analysis of cell-mediated cytotoxicity (CMC) assays. The core function of PAdTrack-CMV is to track and quantify the interaction between effector and target cells, providing researchers with valuable insights into immune cell-mediated killing mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Adeasy adenoviral vector system, by Agilent Technologies (2 mentions)
Bj5183 cells, by Agilent Technologies (1 mentions)
L type triglyceride m kit, by Fujifilm (1 mentions)
Adeasy system, by Agilent Technologies (1 mentions)
P24 elisa kit, by Cell Biolabs (1 mentions)
Dna sequencing, by Sangon (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Adeno x rapid titer kit, by BD (1 mentions)
7 protocols using padtrack cmv
1
Lipid Extraction and Quantification
2
Adenoviral Transduction of SAR1A/B
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Recombinant adenoviruses expressing mouse SAR1A or SAR1B were constructed using pAdTrack-CMV and the AdEasy adenoviral vector system (Agilent Technologies, Lexington MA). Adenoviruses were amplified in Ad293 cells and purified using CsCl gradient ultracentrifugation. Mice were transduced with Ad-GFP, Ad-SAR1A, or Ad-SAR1B via tail vein injection at 0.1 OD per mouse (low dose) or 0.3 OD per mouse (high dose), as described previously (36 (link)). Plasma cholesterol concentrations were measured prior to and 3 or 7 days after transduction as described above.
3
Adenoviral Transduction of SAR1A/B
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Recombinant adenoviruses expressing mouse SAR1A or SAR1B were constructed using pAdTrack-CMV and the AdEasy adenoviral vector system (Agilent Technologies, Lexington MA). Adenoviruses were amplified in Ad293 cells and purified using CsCl gradient ultracentrifugation. Mice were transduced with Ad-GFP, Ad-SAR1A, or Ad-SAR1B via tail vein injection at 0.1 OD per mouse (low dose) or 0.3 OD per mouse (high dose), as described previously (36 (link)). Plasma cholesterol concentrations were measured prior to and 3 or 7 days after transduction as described above.
4
Adenovirus Expressing ESRRG Variant
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Adenovirus expressing the human ESRRG variant1 (NM_001438.3) with a FLAG tag was generated with pAdTrack-CMV and the AdEasy system (Agilent Technologies) as previously described (5 (link)).
5
Recombinant Adenoviral Srx-1 Expression
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The full length of rat Srx-1 cDNA fragments was amplified and then was sub-cloned into the adenoviral shuttle plasmid pAdTrack-CMV (Agilent) containing green fluorescent protein (GFP). Then, the recombinant pAdTrack-CMV-Srx-1-GFP was homologously recombinated with the adenoviral backbone vector pAdEasy-1 in Escherichia coli strain BJ5183. Insert orientation was assessed by DNA sequencing (Sangon). The obtained recombinant plasmids were transfected in HEK293T cells (A.T.C.C.) to generate the recombinant Ad-Srx-1 adenovirus using Lipofectamine 2000 (Invitrogen). After large-scale virus propagation in 293T cells, virus were purified by banding twice on CsCl gradients. The virus titers were determined using p24 ELISA kit (Cell Biolabs).
6
Constructing Adenoviral Vector for ZEB1
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Establishment of the adenovirus (Ad) vector. The ZEB1 fragment was amplified and inserted into the overexpression vector pcDNA 3.1 (promoter CMV; Invitrogen; Thermo Fisher Scientific, Inc.) to construct pcDNA-ZEB1. Thereafter, the fragment was sub-cloned into the Ad vector pAdTrack-CMV (promoter CMV; Agilent Technologies, Inc.) to construct Ad-ZEB1. The empty pAdTrack-CMV vector was used as a negative control (Ad-NC).
7
Generating Recombinant Adenovirus Expressing Netrin-1
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To construct a recombinant adenovirus vector expressing netrin-1, full-length cDNA of human netrin-1 was inserted into pAdTrack-CMV (Agilent) plasmidcontaining green fluorescent protein (GFP). After homologous recombination with pAdEasy-1 in BJ5183, E. coli strain, DNA sequencing was carried out to evaluate the insert identity and orientation by Sangon Company. The Ad-netrin-1 vectors were then transfected into HEK293T cells (ATCC). After propagation in 293T cells, the amplified virus was purified using CsCl2 gradient centrifugation. Then, the viral titer was assessed using the Adeno-X Rapid Titer kit (BD Biosciences, San Jose, CA, USA). For transduction, HUVECs were infected with the recombinant virus suspension of Ad-Srx-1, or Ad-GFP, at the titer of 1 × 10 9 TU/mL for 48 h at 37°C.
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