We determine the optimal seeding density (70% confluence) of your cell line of interest, and optimal antibiotic concentration (>90% cell death after 10 days) prior to transfection.

For transfection, a delivered DNA construct is added to a cell line of interest in combination with Lipofectamine 3000 (optimized with different amounts of DNA and Lipofectamine). We then culture transfected cells in a flask in the presence of antibiotics to select transfected cell pools.

Knock-down is validated by immunoblotting. Transfection efficiency is determined via flow cytometry by staining the cells with an antibody targeting the respective protein, either on the surface or intracellularly.

The cell lines with the highest protein expression are chosen for subsequent experiments (after single cell cloning upon request).