Lentiviruses were prepared using LeGO-iG2 vectors and the Lenti-X packaging system (Takara Bio) and titered with the Lenti-X GoStix Plus (Takara Bio). Cord blood samples were collected from term infants during vaginal or cesarean deliveries. Mononuclear cells were isolated by density-gradient centrifugation on Ficoll-Paque Plus. CD34+ cells were purified using the Indirect CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s protocol and cultured in StemPro-34 SFM medium (Thermo Fisher Scientific) supplemented with 100 ng/mL of thrombopoietin, stem cell factor, and Fms-related tyrosine kinase 3 ligand. After pre-stimulation for 18 h at 37 °C, 2 × 105 cells were seeded in non-tissue culture-treated six-well plates pre-coated with 50 μg/mL of retronectin and transduced with lentiviruses for 48 h at a multiplicity of infection of 20. Transduced cells (GFP-positive) were purified by fluorescence-activated cell sorting using the BD FACSAria Fusion cell sorter. About 1000 sorted cells were mixed with the MethoCult methylcellulose-based medium (H4434, STEMCELL Technologies) and colonies were enumerated after 14 days of culture.
Lenti x packaging system
Manufactured by Takara Bio
Sourced in Japan, United States
The Lenti-X Packaging System is a laboratory equipment designed for the production of lentiviral particles. It provides the essential components required for the efficient packaging of lentiviral vectors.
Automatically generated - may contain errors
Lab products found in correlation
Lenti x gostix plus, by Takara Bio (1 mentions)
Stempro 34 sfm medium, by Thermo Fisher Scientific (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Indirect cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Methocult methylcellulose based medium, by STEMCELL (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Plenti6 v5 d topo vector, by Thermo Fisher Scientific (1 mentions)
Vx 809, by Adooq (1 mentions)
Vx 661, by Adooq (1 mentions)
Vx 445, by Selleck Chemicals (1 mentions)
Dimethyl sulfoxide (dmso), by Selleck Chemicals (1 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions)
Centricon plus 70 filter, by Merck Group (1 mentions)
Recombinant human il 2, by R&D Systems (1 mentions)
Protamine sulfate, by Merck Group (1 mentions)
Pcdh puro bcl xl, by Addgene (1 mentions)
Lenti x 293t, by Addgene (1 mentions)
7 protocols using lenti x packaging system
1
Lentiviral Transduction of CD34+ Cells
2
Differentiation and Genetic Manipulation of 3T3-L1 Adipocytes
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The 3T3-L1 preadipocytes (fibroblasts) were cultured and differentiated into adipocytes as previously described (Zeigerer et al., 2002 (link)). The cells were originally received from G. Baldini (University of Arkansas) in 1992. These cells were last tested for mycoplasma in 2017. Experiments were performed on day 5 after differentiation. The 3T3-L1 adipocyte cell lines stably expressing shRNA sequences against RAB10 or TBC1D4 and expressing control shRNA sequences that do not target genes in the mouse genome have been described previously (Eguez et al., 2005 (link); Sano et al., 2007 (link)).
Immunoabsorption experiments were performed using 3T3-L1 cell lines stably expressing WT and mutant HA-GLUT4-GFP. To generate these cell lines, cDNA constructs encoding WT, F5Y, and F5A-HA-GLUT4-GFP (Lampson et al., 2000 (link); Blot and McGraw, 2006 (link)) were subcloned into the pLenti6/V5-D-TOPO vector (K4955-10; Life Technologies). The 293FT packaging cells were transfected with lentiviral cDNA using Lenti-X packaging system (631276; Takara). Cultured media containing lentiviral particles were harvested after 72 h and used to infect 3T3-L1 preadipocytes. HA-GLUT4-GFP-positive cells were sorted by FACS and cultured in selection medium supplemented with blasticidin (A11139-03; Invitrogen).
Immunoabsorption experiments were performed using 3T3-L1 cell lines stably expressing WT and mutant HA-GLUT4-GFP. To generate these cell lines, cDNA constructs encoding WT, F5Y, and F5A-HA-GLUT4-GFP (Lampson et al., 2000 (link); Blot and McGraw, 2006 (link)) were subcloned into the pLenti6/V5-D-TOPO vector (K4955-10; Life Technologies). The 293FT packaging cells were transfected with lentiviral cDNA using Lenti-X packaging system (631276; Takara). Cultured media containing lentiviral particles were harvested after 72 h and used to infect 3T3-L1 preadipocytes. HA-GLUT4-GFP-positive cells were sorted by FACS and cultured in selection medium supplemented with blasticidin (A11139-03; Invitrogen).
3
Generating CFBE Cell Lines for CFTR Mutant Studies
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The human CF bronchial epithelial cell line CFBE41o- (CFBE), with a CFTR∆F508/∆F508 genotype, was obtained from D. Gruenert (University of California, San Francisco) and grown as reported previously [43 (link)]. CFBE Tet-on cells stably expressing HBH-WT-CFTR-3HA, HBH-R75Q-CFTR-3HA, or HBH-M470V-CFTR-3HA under a tetracycline-responsive promoter were generated by lentivirus transduction under puromycin (3 µg/mL) selection as reported previously [43 (link)]. Briefly, lentiviral particles expressing HBH-WT-, M470V-, or M470V-CFTR-3HA were produced in the HEK293T cells with the Lenti-X Packaging System (Takara Bio, Japan) following the manufacturer’s instructions. The cell lines were generated by transduction with lentiviral particles containing the inducible HBH-CFTR-3HA, followed by selection with puromycin for 2 weeks.
Cadmium nitrate tetrahydrate (Cd), pyocyanin (PYO), and doxycycline hydrochloride (Dox) were purchased from FUJIFILM Wako Chemicals (Japan). VX-809, VX-661, and VX-770 were purchased from AdooQ BioScience (Irvine, CA, USA). VX-445 and DMSO were purchased from Selleck Chemicals (Houston, TX, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.
Cadmium nitrate tetrahydrate (Cd), pyocyanin (PYO), and doxycycline hydrochloride (Dox) were purchased from FUJIFILM Wako Chemicals (Japan). VX-809, VX-661, and VX-770 were purchased from AdooQ BioScience (Irvine, CA, USA). VX-445 and DMSO were purchased from Selleck Chemicals (Houston, TX, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.
4
Lentiviral Overexpression of TKT in H1299 Cells
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TKT was ectopically overexpressed in H1299 cells using Precision LentiORF TKT with stop codon (clone ID: PLOHS_100005708) or red fluorescent protein (RFP) as a control. Viral particles were produced by transfecting HEK293T cells using the Lenti-X Packaging System (Clontech Laboratories) as per manufacturer’s directions. Subsequently, target cell lines were transduced with viral supernatants in the presence of 8 ng/µL polybrene. Transduced cells were collected via cytometric sorting (BD Fusion3, BD Bioscience). Expression was confirmed by RT-qPCR and western blotting.
5
Generating Stable IGF2BP1-Expressing DEE Cells
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The HEK 293T cells were seeded in the 6-well plate and cultured until the cells reached 60% confluence. Lentiviral particles were produced in the HEK293T cells with the Lenti-X Packaging System (Clontech) following the manufacturer’s instructions. Briefly, 37 µL of Lenti-X HT Packaging Mix, 6 µL (about 4.0 µg) of pLVX-Tight-Puro-IGF2BP1 plasmids, and 557 µL of Xfect Reaction Buffer were put together and then mixed with 7.5 µL of Xfect Polymer diluted in 592.5 µL of Xfect Reaction Buffer. The mixture was gently mixed and then incubated for 10 min at 25 °C, followed by addition to the 6-well plate. At 10 hpt, the mixture was replaced by DMEM supplemented with 10% FBS. At 72 hpt, the supernatant was collected to infect DEE cells. At 24 hpi, the DEE cells were cultured in DMEM supplemented with 5% fetal bovine serum, 500 μg/mL G418, 2 μg/mL puromycin, and 100 µg/mL of streptomycin sulfate. After approximately 17 days, the DEE cell line that stably expressed IGF2BP1 was established.
6
Lentiviral Transduction of CAR T Cells
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DNA encoding CARs were inserted into the pLVX-EF1α-IRES-Puro lentiviral transfer vector (Clontech). Lentiviral vectors were packaged using 293T cells with the Lenti-X packaging system (Clontech) and concentrated by ultrafiltration using Centricon Plus-70 filters (EMD Millipore) at 1,500 rpm for 2 h at 15°C. Jurkat T cells were transduced with lentiviral vectors using spinoculation in a 48-well tissue culture plate. The plate was spun at 2,500 rpm for 90 min at 32°C. The transduced cells were selected in complete RPMI medium containing 0.25 μg/ml puromycin. To transduce primary human CD8+ T cells, T cells were stimulated with human T-activator CD3/CD28 Dynabeads (Life Technologies) at a 3:1 bead to cell ratio for 1 day, followed by spinoculation in the presence of 10 μg/ml protamine sulfate (Sigma-Aldrich). Beads were removed 2 days later, and the cells were cultured and expanded in complete RPMI medium containing 300 IU/ml recombinant human IL2 (R&D Systems) for 3–5 additional days before being used for experiments.
7
Overexpression of Bcl-2 and Bcl-xL in MCF10A cells
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Overexpression of Bcl-2 and Bcl-xL in MCF10A cells was performed by lentiviral transduction using the Lenti-X Packaging System (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The pCDH-puro-Bcl2 plasmid (Addgene plasmid #46971) and pCDH-puro-Bcl-xL (Addgene plasmid #46972) were added into supplied nanoparticle complexes for 10 min and applied to Lenti-X 293T cells to produce the virus. The medium was changed after 24 h and viral supernatant was harvested after 48 h, filtered, and used to infect cells at an approximate MOI of 10 along with 1 µg/mL Polybrene. The plate was then immediately centrifuged for 1.5 h at 1000 rpm. Cells were selected with 1 µg/mL puromycin at 2 days after infections and maintained in puromycin-containing media. Puromycin was removed from the medium for experimental conditions. pCDH-puro-Bcl2 and pCDH-puro-Bcl-xL were gifts from Jialiang Wang [36 (link)].
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