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

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==Entry title==
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==03/01/13==
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* Insert content here...
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HEK293 Luc #4 DAPI staining
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Assay on 96-well plate
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 +
 
 +
 
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----
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* DAPI/Hoescht: made by Dr. Haynes; 5000x in sterile dH<sub>2</sub>O
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* Diluted Hoescht to working concentration (1x) in 1xPBS.  
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* Added 2 mL to each well
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* Let sit at room temperature for 5, 10, 20, and 30 mins in the dark to develop signal
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 +
 
 +
----
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Results:
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* 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
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** takes longer time to grow
 +
** often wells turned out empty or cells died
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** but a few very good nicely formed colonies
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 +
 
 +
----
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Literature review results:
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* DAPI doesn't stain replicating DNA, yet it's one of the best nuclei staining dyes.
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* luciferin signal is only detectable with very sensitive liquid gas cooled cameras
 +
 
 +
----
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Next steps:
 +
* Detect luciferin signal with antibody staining instead of direct bioluminescence
 +
* Therefore fixing cells with formaldehyde
 +
* Should improve DAPI signal

Revision as of 23:43, 18 March 2013

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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



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