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

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08/06/10

 * &#x2713; Senescence assay: C12-FDG treatment and flow cytometry
 * &#x2713; H3me reporter lines: expand KAH142 for freezing; split others for another transfection test
 * &#x2713; 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 Note: for the Western and microscopy, add dox the day before processing samples
 * KAH130-
 * KAH131-


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