Silver: FKHR Translocation Assay

Day 1

 * 1) Plate 786-O cells onto a 384-well clear-bottom plate
 * 2) *~4000 cells/well in 50 µl of complete media (DMEM/10% Fetal Clone)
 * 3) *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

 * 1) Resuspend Compounds in serum-free media to correct dilution, remove media from cells, and then add compound dilution to cells.
 * 2) *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.
 * 3) Incubate at 37 degrees C, 5%CO2 for 1h.
 * 4) Remove media
 * 5) Fix cells in 3.7% Formaldehyde in PBS for 10 min. at 4 degrees C or RT.
 * 6) Wash cells 2-3 times with PBS.
 * 7) Incubate cells with M5 anti-FLAG antibody (Sigma) at 1:1000 dilution in 5%FBS/0.2%TX100/PBS for 1h at RT.
 * 8) Remove primary antibody and wash cells 2-3 times with PBS.
 * 9) 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.
 * 10) Remove antibody and Hoechst and wash cells 2-3 times in PBS.
 * 11) Keep cell sin PBS for storage and imaging. Store at 4 degrees C.