Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA241941

Local Sample ID:	F2-27-09
Subject ID:	SU002502
Subject Type:	Cultured cells
Subject Species:	Homo sapiens

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Treatment:

Treatment ID:	TR002514
Treatment Summary:	Transduction All cells were transduced at the indicated multiplicity of infection (MOI) as calculated by titration of vector stocks on 293T cells and expressed as transducing units (TU)/293T cell. Cell lines were transduced with LV for 16 hours, washed and kept in culture until reading transduction efficiency by flow cytometry. Human CB-derived HSPC were cultured in serum-free StemSpan medium (StemCell Technologies) supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), 100 ng/ml recombinant human stem cell factor (rhSCF), 20 ng/ml recombinant human thrombopoietin (rhTPO), 100 ng/ml recombinant human Flt3 ligand (rhFlt3), and 20 ng/ml recombinant human IL6 (rhIL6) (all from Peprotech) 16 to 24 hours prior to transduction. HSPC were then transduced at a concentration of 1×106 cells per milliliter with VSV-gp-pseudotyped SINLV for 16 hours at the indicated multiplicity of infection (MOI) in the same medium. G-CSF mobilized peripheral blood CD34+ cells were maintained in culture in CellGro medium (Cell Genix) containing a cocktail of cytokines: 60 ng/ml IL-3, 100 ng/ml TPO, 300 ng/ml SCF, and 300 ng/ml FLT-3L (all from Cell Peprotech). Transductions were performed with the indicated dose of vectors for 16 hours in the same cytokine-containing medium. For single hit reporter LV transductions cells were washed and maintained in serum-free medium supplemented with cytokines as above until the reading of the percentage of positive cells by FACS. To perform OE, KD and KO experiments cells were transduced with the respective LV and then challenged with a second transduction with or without drugs. Except for primary cells, KD and KO cells were selected with Puromycin before the second hit of transduction.

Treatment ID:

TR002514

Treatment Summary:

Transduction All cells were transduced at the indicated multiplicity of infection (MOI) as calculated by titration of vector stocks on 293T cells and expressed as transducing units (TU)/293T cell. Cell lines were transduced with LV for 16 hours, washed and kept in culture until reading transduction efficiency by flow cytometry. Human CB-derived HSPC were cultured in serum-free StemSpan medium (StemCell Technologies) supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), 100 ng/ml recombinant human stem cell factor (rhSCF), 20 ng/ml recombinant human thrombopoietin (rhTPO), 100 ng/ml recombinant human Flt3 ligand (rhFlt3), and 20 ng/ml recombinant human IL6 (rhIL6) (all from Peprotech) 16 to 24 hours prior to transduction. HSPC were then transduced at a concentration of 1×106 cells per milliliter with VSV-gp-pseudotyped SINLV for 16 hours at the indicated multiplicity of infection (MOI) in the same medium. G-CSF mobilized peripheral blood CD34+ cells were maintained in culture in CellGro medium (Cell Genix) containing a cocktail of cytokines: 60 ng/ml IL-3, 100 ng/ml TPO, 300 ng/ml SCF, and 300 ng/ml FLT-3L (all from Cell Peprotech). Transductions were performed with the indicated dose of vectors for 16 hours in the same cytokine-containing medium. For single hit reporter LV transductions cells were washed and maintained in serum-free medium supplemented with cytokines as above until the reading of the percentage of positive cells by FACS. To perform OE, KD and KO experiments cells were transduced with the respective LV and then challenged with a second transduction with or without drugs. Except for primary cells, KD and KO cells were selected with Puromycin before the second hit of transduction.