Cell Transfection Sample Clauses

Cell Transfection. For ps20 purification: Transfection of 293F cells in 30ml conical flasks 1µg of pBK-WFDC1 was combined with 1ug of PEI or Lipofectamine in 100µl of optimum™ and incubated for 20mins. 1µg of DNA was used per 106 cells. The mixture was added to cells drop wise and cells were cultured for different time points to express protein as required. Alternatively, 293T cells were transfected in 72cm2 plates. 1ug DNA /106 cells was used and media was changed to DMEM/F-12 after 6 hrs. Cells were cultured to express protein for maximum 48h as 293T cell viability decreases rapidly in serum free media. For the transient expression of ps20 protein species in 293T cells to generate conditioned media (CM) a multiwell transfection assay was optimised using the GeneIn® reagent. The afternoon prior to the day of transfection cells were seeded in 6 well plates at 4x105/well. The following day, media was replaced. The transfection reagents were prepared for each well according to the schema presented in table 2.2 and incubated together at room temperature for 15 mins. 200μl of each mixture was then added dropwise to 293T cells. CM was collected after 48h, clarified, filtered and stored at -80⁰C. Where cells where transfected in larger vessels all experimental components including, volume of media added, number of cells seeded, and quantities of transfection agents used, were scaled up in proportion to the surface area of the vessel used. Table 2.2 Preparation of GeneIn™ for transfection optimum to 200μl plasmid DNA 2μg Blue reagent 8μl Red reagent 4μl