User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/03/01

From OpenWetWare

< User:David Dreher | Notebook | Chromatin controlled cell pattern | 2013 | 03(Difference between revisions)
Jump to: navigation, search
(Entry title)
Current revision (23:43, 18 March 2013) (view source)
(03/01/13)
 
Line 8: Line 8:
==03/01/13==
==03/01/13==
HEK293 Luc #4 DAPI staining
HEK293 Luc #4 DAPI staining
 +
Assay on 96-well plate
Assay on 96-well plate

Current revision

Project name Main project page
Previous entry      Next entry

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



Personal tools