The genes of ROR-1 CARs comprising of a single chain variable region (scFv) from Zilovertamab, a short IgG4 Fc hinge or a long IgG4 Fc hinge-CH2-CH3 spacer, CD28 transmembrane domain, and an intracellular signaling domain contains 4-1BB-CD3zeta were synthesized by Genewiz (Suzhou, Jiangsu, China). The genes were cloned into pALD-LentiEGFP vectors (Aldevron) by recombinant cloning via Apa1/Nhe1 cloning sites using Hyper Assembly Cloning Kit (APEXBIO Tech, Houston, TX, USA) following the protocol. The GFP marker was removed from this cloning process. Lentivirus were prepared in HEK293T-cells by transduction with a four-plasmid system coding for the lentivector genomes, pALD-VSV-G, pALD-Rev, and pALD-GagPol (Aldevron) using Lipofectamin 3000 (Invitrogen). The lentivirus was harvested at 48 and 72 h after transduction and concentrated by an Amicon Ultra-15 100 kDa MWCO centrifugal concentrator (MilliporeSigma, MA, USA) if necessary. All the virus preparations were frozen at −80 °C for further experimentation.
Amicon ultra 15 100 kda mwco centrifugal concentrator
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra-15 100 kDa MWCO centrifugal concentrator is a laboratory device used for the concentration of macromolecules, such as proteins and enzymes, from a sample solution. It utilizes a semi-permeable membrane with a molecular weight cut-off of 100 kilodaltons to selectively retain the desired macromolecules while allowing smaller molecules and solvents to pass through during a centrifugation process.
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Lab products found in correlation
Pald gag pol, by Aldevron (1 mentions)
Pald vsvg, by Aldevron (1 mentions)
Pald rev, by Aldevron (1 mentions)
Pald lentiegfp vectors, by Aldevron (1 mentions)
Lipofectamin 3000, by Thermo Fisher Scientific (1 mentions)
Gravity flow column, by Bio-Rad (1 mentions)
Expressplus page gel, by GenScript (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
2 protocols using amicon ultra 15 100 kda mwco centrifugal concentrator
1
Generation of ROR-1 CAR Lentivirus
2
Ni-NTA Affinity Purification of Recombinant Proteins
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All purification steps are performed on the bench at RT. INDIGO‐Ni agarose resin (Cube Biotech) was rinsed with wash buffer (20 mM Tris, pH 8.0, 1 M NaCl), added to the media, and stirred at RT for 3 hr. After stirring, the resin was allowed to settle for 30 min. The media was then decanted and disposed of, and the resin suspended in wash buffer. The suspended resin was added to 30‐ml gravity‐flow columns (BIO‐RAD) and washed with 10 CV of wash buffer. The column was washed with 3 CV of wash buffer containing 20, 40, 60, 80, and 100 mM imidazole were performed sequentially. The imidazole stock solution (1 M) was adjusted to pH 8 with NaOH before use. The protein was eluted with wash buffer containing 500 mM imidazole. Washes and elutions were concentrated using an AMICON Ultra‐15100 kDa MWCO centrifugal concentrator (regenerated cellulose membrane, Millipore Sigma) and run on ExpressPlus PAGE gels (GenScript) and stained with SimplyBlue SafeStain (Novex) to confirm the location of the eluted protein. Protein concentration was determined by A280 using calculated extinction coefficients based on the amino acid sequence.
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