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Found 1 matching solution for this experiment
pMT-hFIX/M
Jafar Vatandoost, Department of Biology, Hakim Sabzevari Univers
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All constructs along with pCoHygro plasmid were transfected into Drosophila S2 cells (Invitrogen, Lot no; 769,915) employing the calcium phosphate co-precipitation method with minor modifications [19]. After 48 h, the cells were cultured in 300 μg of hygromycin B/ml. To select the clones stably expressing FIX, the single cell strategy was employed [20]. |
To assess the functional rFIX expression, stable S2 cells were induced with 0.5 mM CuSO4 in the presence of 6 μg/ml of vitamin K1. Total FIX expression was quantified in conditioned media employing a FIX-specific antigen assay (ELISA) following the manufacturer’s instructions (Asserachrom, hFIX: Ag). The FIX clotting activity was determined employing a modified one-stage aPTT assay specific for FIX as described previously [18]. |
Protocol tips |
All constructs along with pCoHygro plasmid were transfected into Drosophila S2 cells (Invitrogen, Lot no; 769,915) employing the calcium phosphate co-precipitation method with minor modifications [19]. After 48 h, the cells were cultured in 300 μg of hygromycin B/ml. To select the clones stably expressing FIX, the single cell strategy was employed [20]. |
Downstream tips |
To assess the functional rFIX expression, stable S2 cells were induced with 0.5 mM CuSO4 in the presence of 6 μg/ml of vitamin K1. Total FIX expression was quantified in conditioned media employing a FIX-specific antigen assay (ELISA) following the manufacturer’s instructions (Asserachrom, hFIX: Ag). The FIX clotting activity was determined employing a modified one-stage aPTT assay specific for FIX as described previously [18]. |
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