03/01/13
HEK293 Luc #4 DAPI staining
Assay on 96-well plate
- DAPI/Hoescht: made by Dr. Haynes; 5000x in sterile dH2O
- Diluted Hoescht to working concentration (1x) in 1xPBS.
- Added 2 mL to each well
- Let sit at room temperature for 5, 10, 20, and 30 mins in the dark to develop signal
Results:
- strong fluorescence signal only in dead/detached cells
- slightly better signal after 5 mins, no increase in signal afterwards
- still too weak signal in living cells
- 96-well plate resulted in few distinct good colonies
- problematic when too close to the wall
- takes longer time to grow
- often wells turned out empty or cells died
- but a few very good nicely formed colonies
Literature review results:
- DAPI doesn't stain replicating DNA, yet it's one of the best nuclei staining dyes.
- luciferin signal is only detectable with very sensitive liquid gas cooled cameras
Next steps:
- Detect luciferin signal with antibody staining instead of direct bioluminescence
- Therefore fixing cells with formaldehyde
- Should improve DAPI signal
|