Silver: FKHR Translocation Assay

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Day 1

  1. 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

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