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The parental murine B16-BL6 melanoma cell line [57, 58] was provided by Dr. Sarah Netzel-Arnett (University of Maryland School of Medicine) and maintained in DMEM (Corning) supplemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine and 1% penicillin-streptomycin (both from Corning). All cells were maintained at 37°C in 5% CO2. Cells were detached with non-enzymatic cell dissociation solution (Sigma-Aldrich) for routine passaging and experiments.Cells were plated, allowed to attach for 5 hr, and then transfected using Lipofectamine RNAiMax transfection reagent (Invitrogen) according to the manufacturers’ instructions with either negative control siRNA, Fn14 siRNA #1 or #4 targeted to the mouse Fn14 transcript (all three siRNAs at a final concentration of 20 nM),. All siRNAs were purchased from Qiagen: negative control (#1022076), mouse Fn14 (#SI01452311, #SI01452332). Cells were harvested at 24 hr post-transfection and seeded onto Matrigel invasion chambers as described above, with the remaining cells harvested at either 24 or 48 hr post-transfection for Western blot analysis. |
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| Protocol tips |
| The parental murine B16-BL6 melanoma cell line [57, 58] was provided by Dr. Sarah Netzel-Arnett (University of Maryland School of Medicine) and maintained in DMEM (Corning) supplemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine and 1% penicillin-streptomycin (both from Corning). All cells were maintained at 37°C in 5% CO2. Cells were detached with non-enzymatic cell dissociation solution (Sigma-Aldrich) for routine passaging and experiments.Cells were plated, allowed to attach for 5 hr, and then transfected using Lipofectamine RNAiMax transfection reagent (Invitrogen) according to the manufacturers’ instructions with either negative control siRNA, Fn14 siRNA #1 or #4 targeted to the mouse Fn14 transcript (all three siRNAs at a final concentration of 20 nM),. All siRNAs were purchased from Qiagen: negative control (#1022076), mouse Fn14 (#SI01452311, #SI01452332). Cells were harvested at 24 hr post-transfection and seeded onto Matrigel invasion chambers as described above, with the remaining cells harvested at either 24 or 48 hr post-transfection for Western blot analysis. |
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