User:Karmella Haynes/Notebook/Polycomb project/2010/08/06

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  • ✓ Senescence assay: C12-FDG treatment and flow cytometry
  • ✓ H3me reporter lines: expand KAH142 for freezing; split others for another transfection test
  • ✓ KAH130, 131 lines: split cells; expand for freezing

Senescence assay

  1. KAH126-1
  2. KAH126-1 dox
  3. KAH126-3
  4. KAH126-3 dox
  5. KAH154-2
  6. KAH154-2 dox
  7. KAH132-8
  8. KAH132-8 dox
  9. FTRx DMSO
  10. FTRx rotenone
  11. FTRx dox
  12. FTRx (no C12-FDG) "blank"

> Treat w/ 1.5 μg/mL C12-FDG/ 37°C/ 45 min. before harvesting for flow cytometry
> Wash pelleted cells twice with DPBS and resuspend in 0.5 mL DPBS (do not spin through filter-cap)

H3me Reporter lines
> Checked transfections for YFP expression again today
--> KAH146-1 showed YFP expression in lots of cells (but not 100%) > Expand KAH142-3, 142-5, and 146-1 for freezing; Keep back-up plate for 142-3 and 146-1
> Make 12-well plates for Fugene transfection with KAH60/pcVN
> Split back-up 6-well stocks (if confluent)

KAH130, 131 lines
> Interesting: Dox induction of Pc-ATF is very toxic after 3-4 days, unlike the controls that lack VP64
> Note: Will have to induce with dox for a shorter time period to do any analysis
> Expand the following (from 6-well plates to 10 cm dishes) and seed new 6-well plates (no ab's) for -/+ dox Western; also set up microscopy 12-well glass-bottom plate

  • KAH130-
  • KAH131-

Note: for the Western and microscopy, add dox the day before processing samples