Dexamethasone Transfection Protocol

Application

Dexamethasone (Millipore cat. no. TR-1002-G) is used to induce glucocorticoid responsive cells. Cells that are transiently or stably transfected with a vector containing the dexamethasone inducible promoter, MMTV, can be induced from no or low level expression to high level expression of the cloned sequence using this protocol.

Specifications

• Storage: Upon receipt, store at –20°C
• Sterility: Reagent is supplied sterile
• Application Reagent: 0.1 mM dexamethasone in 100% ethanol 1000X stock solution

Protocol

1. Induction of stable transfected clones: at least 10 clones should be isolated, expanded and tested for level of induction. Levels of induction can range from no increase in expression over uninduced cells to an induction level two orders of magnitude over the uninduced cells. The level of induction can be measured by directly assaying the product of the cloned sequence or by northern blot analysis of the levels of mRNA transcribed from the cloned sequence.
2. Plate cells at approximately 40% confluence, in duplicate, 24 hours before induction.
3. Remove medium and add fresh, complete growth medium containing 0.1 µM dexamethasone (final concentration) to the induced plate, and fresh growth medium only to the uninduced plate.
4. Incubate both plates at 37°C for 16 to 48 hours before harvesting.
5. Induction of cells transiently transfected: add 0.1 µM dexamethasone (final concentration) to the complete growth medium 16 to 24 hours before harvesting.

References

1. Lee, F. et al., Nature 1981; 294:228
2. Lee, F. et al., Nucleic Acids Research 1984; 12:4191
3. Morley, K.L. et al., Nucleic Acids Research 1987; 15:6973