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02/23/13
- DAPI/ luciferase staining
- New cell plates
DAPI/ luciferase staining
- D-luciferin stock solution: 30mg/ml in 1xPBS, working solution is 150ug/ml (200x)
- Hoescht: made by Dr. Haynes; 5000x in sterile dH2O
- Diluted Hoescht and D-luc to working concentration (1x) in 1xPBS. Added 2 mL to each well...
Used 3 wells:
- DAPI + D-luc
- DAPI only
- PBS
- Let cells sit at room temperature for 10 minutes in the dark to develop signal
Results/ Conclusions:
- 2 mL medium is susceptible to drying at outer wells; disrupts cell growth
- Did not detect D-luc signal. Perhaps D-luc imaging should start immediately after adding reagents. The D-luc signal has a short half-life.
- Cells rounded up a bit after sitting in PBS. Next time use 10% FBS in PBS, or try DMEM without red dye.
- DAPI signal was barely visible
Note: add photos here
New cell plates
Dr. Haynes; For more practice staining (Monday)
- HEK293 luc #4, 6-well plate, ~1x105 cells/ well, 4 mL growth medium
- HEK293 Gal4-EED, 6-well plate, ~1x105 cells/ well, 4 mL growth medium
Stock cultures (T75 flasks)
- HEK293 luc #4, 1:5
- HEK293 Gal4-EED, 1:4
- HEK293 Gal4-EED, 1:4 in 0.5 μg/mL puromycin (to select for Gal4-EED RNAi tuner)
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