Stable transfection

1. Test untransfected cells to determine the concentration of antibiotic(s) that kills 99% of cells within 7 to 10 days. For example, 5 mg/ml blasticidin is sufficient for HEK293. When used in combination, antibiotic concentrations should be reduced. For example, 2.5 mg/ml blasticidin plus 150 mg/ml neomycin is sufficient for HEK293. Proceed with the transient transfection protocol given earlier, using two 10-cm dishes for each reporter. Following overnight incubation, remove transfection complexes and add fresh medium. After 24 h, and from this point on, add medium containing the selected concentration of antibiotic(s). Change the medium every 2 days.

2. After 10 days, trypsinize the cells and, using one of the dishes, test reporter activity. From the remaining dish, count the cells and seed into a 96-well plate at 1 cell per well.

3. Allow cells to grow until single colonies appear («7 days). When colonies have grown to 30 to 50% confluence («2 weeks), trypsinize and transfer to 24-well plates. When cells reach 50% confluence («4 weeks), transfer to 6-well plates. Sufficient cells are now present to allow identification of which clones have reporter activity and continue expanding them. Promising clones should be stored in liquid nitrogen as the selection process continues.

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