Silver: FKHR Translocation Assay
- Plate 786-O cells onto a 384-well clear-bottom plate
- ~4000 cells/well in 50 µl of complete media (DMEM/10% Fetal Clone)
- If also infecting, infect cells by adding AdFKHR virus at 1:10,000 dilution to cells in suspension before plating. Let infection go 19-24 hours.
Day 2 - Compound Treatment
- Resuspend Compounds in serum-free media to correct dilution, remove media from cells, and then add compound dilution to cells.
- If doing serial dilutions, start with 100 µl of highest dilution and serially dilute 1:2 by adding 50 µl of previous dilution to 50 µl of media. Can perform serial dilutions right on cells or on empty plate to transfer to cells.
- Incubate at 37 degrees C, 5%CO2 for 1h.
- Remove media
- Fix cells in 3.7% Formaldehyde in PBS for 10 min. at 4 degrees C or RT.
- Wash cells 2-3 times with PBS.
- Incubate cells with M5 anti-FLAG antibody (Sigma) at 1:1000 dilution in 5%FBS/0.2%TX100/PBS for 1h at RT.
- Remove primary antibody and wash cells 2-3 times with PBS.
- Incubate cells with secondary antibody and Hoechst 33258 or DAPI (eg. AlexaFluor-594 goat anti-mouse at 1:1000) in 5%FBS/0.2%TX100/PBS for 1h at RT.
- Remove antibody and Hoechst and wash cells 2-3 times in PBS.
- Keep cell sin PBS for storage and imaging. Store at 4 degrees C.