User:Karmella Haynes/Notebook/Polycomb project/2010/06/28

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06/28/10

  • ✓ Senescence assay: C12-FDG treatment & flow cytometry



Senescence assay
> Samples (~3.5 mL medium/ well, plated on 6/24/20):

  1. KAH126-1
  2. KAH126-1 +1 μg/mL dox
  3. KAH126-3
  4. KAH126-3 +1 μg/mL dox
  5. KAH132-8
  6. KAH132-8 +1 μg/mL dox
  7. KAH154-2
  8. KAH154-2 +1 μg/mL dox
  9. FTRx + 0 rotenone (7.0 μL DMSO)
  10. FTRx + 0.1 μg/mL rotenone (1.75 μL/ 5.25 μL DMSO)
  11. FTRx + 0.2 μg/mL rotenone (3.5 μL/ 3.5 μL DMSO)
  12. FTRx + 0.4 μg/mL rotenone (7.0 μL/ 0 μL DMSO)
  13. FTRx blank

> Note: added fresh dox to "dox" samples on 6/27
> C12-FDG: add 30 μg/mL; 37°C/ 1 hr.; harvest and strain cells for flow cytometry (1x106 cells/ 500 μL)
> Flow cytometry: RFP and FITC channels, count 10,000 live cells per sample

> Results:

  • Background signal too high (104); titer C12-FDG and rotenone to lower concentrations for FTRx cells
  • Induction of RFP successful for all Pc-ATF cells. Off-state nearly equal to FTRx untransfected cells. 126-1, 126-3 show a sub-population of non-induced cells after dox treatment.


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