User:Karmella Haynes/Notebook/Polycomb project/2010/07/03

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07/03/10

  • ✓ Senescence assay (optimization): flow cytometry for C12-FDG titration
  • ✓ Senescence assay: set up 6-well plates for 126-1, 126-3, 132-8, 154-2 -/+ dox; FTRx DMSO/rotenone
  • ✓ Transformation prep: FTRx 6-well (new stable lines for KAH130, 131)
  • ✓ Cell culture: split confluent stock cultures
  • ✓ RT-PCR cultures: set up 6-well plates (-/+ dox samples) to re-do RT-PCR (next time use less RNA for cDNA synth); 126-1, 126-3, 132-8, 154-2, FTRx
  • ✓ p16INK western/RT-PCR: FTRx 6-well -/+ 0.2 μg/mL rotenone; FTRx 10 cm plate -/+ μg/mL rotenone



Senescence assay optimization
> Flow cytometry cell samples
1-6. Plate 1: Untreated (DMSO)
7-12. Plate 2: 0.2 μg/mL rotenone (3 days)
blank: FRTX cells (stock culture), no C12-FDG

> C12-FDG treatment (37°C/ 45 min.)
--> use 1/10 serial dilution
1,7. 0 C12-FDG/ 30 μL DMSO
2,8. 0.0015 μg/mL C12-FDG/ 30 μL DMSO
3,9. 0.015 μg/mL C12-FDG/ 30 μL DMSO
4,10. 0.15 μg/mL C12-FDG/ 30 μL DMSO
5,11. 1.5 μg/mL C12-FDG/ 30 μL DMSO
6,12. 15.0 μg/mL C12-FDG/ 30 μL DMSO

> Results: Could see slight but subjectively significant increase in FITC signal for rotenone treated cells for 0.15, 1.5, and 15.0 μg/mL C12-FDG samples. Background signal increases orders of magnitude with C12-FDG concentration.
> Next: Set up dox induction of Pc-ATF lines and FTRx rotenone treatment (4 days) for next C12-FDG assay. Will determine whether RFP signal is associated with senescence (high FITC)



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