Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4). Lentiviral particles containing these vectors were isolated using the Lenti-vpak Lentiviral Packaging Kit (Origene, TR30037) as directed by the manufacturer. When ready to transduce, lentivirus was thawed rapidly at 37 °C. Cells were seeded 50,000 cells in 1 mL maintenance media without any antibiotics into 6 well plates and reverse transduced with 500 μL of virus per well. Control wells were seeded in the absence of virus. After 48 h, cell lines were selected with puromycin at the following doses: MCF-10A .4 μg/mL for selection and maintenance, all cells grown in DMEM were selected at 2 μg/mL and maintained at 0.5 μg/mL, and all cells grown in RPMI were selected at 0.5 μg/mL and maintained at 0.25 μg/mL. After selection, cells were then flow-sorted at the Vanderbilt Flow Cytometry Shared Resource on the FACSAria III (BD) for the top 1% of GFP expression from the baseline cell pools and utilized in indicated experiments.
Ps100093
Manufactured by OriGene
The PS100093 is a laboratory equipment product. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
4 protocols using ps100093
1
Lentiviral Transduction of GFP-Tagged Proteins
2
Lentiviral Transduction of HEK293T Cells
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In separate 15 mL conicals, 1 μg of expression clone cDNA (Origene NM_001923) or control cDNA (Origene PS100093) was mixed with packaging plasmids MD2G (1 μg, Addgene 12,259) and PSPAX2 (1 μg, Addgene 12,260) in 600 μL per plate OPTIMEM and Lipofectamine 2000 transfection reagent (Invitrogen 11668027) was incubated with an equal volume of OPTIMEM (1:30 v/v) for 5 min prior to tubes being combined and incubated for 40 min at room temperature. The DNA-Lipofectamine mix was diluted with 8 mL of DMEM and added to HEK293T cells at 40% confluency in 10 cm plates. The next day, media was replaced with 6 mL fresh DMEM for 24 h.
For each control or knock-down clone, media was removed from HEK293T cells, filtered through a 0.45-micron syringe filter, mixed with 10 μL polybrene transfection reagent, and added to new HEK293T cells at 50% confluency. The original HEK293T media was replaced with 6 mL fresh DMEM for 24 h and the infection process was repeated. 24 h after the second infection, the new HEK293T infection media was removed, and cells were seeded for Western blot analysis.
For each control or knock-down clone, media was removed from HEK293T cells, filtered through a 0.45-micron syringe filter, mixed with 10 μL polybrene transfection reagent, and added to new HEK293T cells at 50% confluency. The original HEK293T media was replaced with 6 mL fresh DMEM for 24 h and the infection process was repeated. 24 h after the second infection, the new HEK293T infection media was removed, and cells were seeded for Western blot analysis.
3
Generating Tat-GFP Expressing Cell Line
4
Lentiviral Transduction of HAP1 Cells
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