Life Technologies
Lipofectamine 2000 Transfection Reagent
11668019
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The aim of my experiment was to deplete NFATc1 at the protein level in endothelial cells. For this, NFATc1 siRNA at different molal concentrations were transfected in cells. Lipofactamine 2000 from Life Technologies was used as transfection reagent. After transfection cells were incubated for 40hrs, proteins were extracted and resolved in 10% SDS-PAGE followed by immunoblotting with specific NFATc1 antibodies.
siRNA transfection
Endothelial cells
Endothelial cells were cultured in a 60mm dish and NFATc1 siRNA at different molal concentrations (100nmole or 200nmole) was transfected using Lipofactamine 2000. Cells transfected with non-target siRNA was used as a control. All steps of transfection were followed as per manufacturer’s instructions. Transfected cells were allowed to incubate for 40hrs to deplete NFATc1 at protein level. Proteins from cells were extracted in RIPA buffer and resolved in 10% SDS-PAGE. Resolved proteins were Western transfer and probed with specific NFATc1 antibodies. ECL detection was used to visualize HRP activity.
NA
Lipofactamine 2000 worked well and showed excellent transfection efficiency. Almost 95% of NFATc1 was depleted in transfected cells as compared to control siRNA transfected cells.
None
High transfection efficiency
Measurable cytotoxicity was observed
Lipofactamine 2000 is a good for siRNA transfection in human cell lines
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