Team:EPF-Lausanne/Protocol/TrypanBlue

From 2012.igem.org

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<noinclude>{{:Team:EPF-Lausanne/Template/Header}}</noinclude>
<noinclude>{{:Team:EPF-Lausanne/Template/Header}}</noinclude>
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{{:Team:EPF-Lausanne/Template/ProtocolHeader| Trypan Blue Method | {{{1|}}}}}
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{{:Team:EPF-Lausanne/Template/ProtocolHeader|Trypan Blue Method | {{{1|}}}}}
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{
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<noinclude>{{:Team:EPF-Lausanne/Template/SetTitle|Trypan Blue}}</noinclude>
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<table>
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<tr>
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<td> Cell density (10^6 ml) </td>
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<td> Dilution</td>
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<td> PBS (µl)</td>
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<td> Cells (µl) </td>
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<td>Trypan Blue (µl) </td>
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</tr>
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<tr>
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<td> 1-2 </td>
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<td> 4 </td>
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<td> 100 </td>
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<td> 50 </td>
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<td> 50 </td>
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</tr>
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<tr>
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<td> 2-4.5 </td>
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<td> 8 </td>
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<td> 125 </td>
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<td> 50 </td>
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</tr>
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<tr>
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<td> 4.5-7 </td>
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<td> 12 </td>
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<td> 120 </td>
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<td> 15 </td>
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<td> 45 </td>
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</tr>
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<tr>
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<td> >7 </td>
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<td> 16 </td>
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<td> 137.5 </td>
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<td> 12.5 </td>
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<td> 50 </td>
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</tr>
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</table>
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=== Sampling ===
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{| class="wikitable" style="text-align: center; color: black;"
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|Cell Density (10^6/ml)
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|Dilution       
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|PBS              (µl)
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|Cells            (µl)
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|Trypan Blue (µl)
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|-
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|1 - 2
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|4
 +
|100
 +
|50
 +
|50
 +
|-
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|2 - 4.5
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|8
 +
|125
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|25
 +
|50
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|-
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|4.5 - 7
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|12
 +
|120
 +
|15
 +
|45
 +
|-
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|> 7
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|16
 +
|137.5
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|12.5
 +
|50
 +
|-
 +
|}
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#Take xµL ofPBS in 96 well plate
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#Add required volume of cell culture. Mix once
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#Take x µL of PBS in a 96-well plate.
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#Bring the plate back to microscope, add trypan blue to the PBS + Cell mixture just before counting the sample.  
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#Add the required volume of cell culture. Mix once.
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      Trypan Blue is toxic to cells. If left too long with trypan blue, even healthy cells will die.  
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#Bring the plate back to the microscope, add Trypan Blue to the PBS + cell mixture just before counting the sample.  
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Trypan Blue is toxic to cells. If left too long with it, even healthy cells will die.
'''Calculation of LCD :'''
'''Calculation of LCD :'''
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LCD = Cell Count/ ( 100* 4) * Dilution
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LCD = Cell Count/(100*4)*Dilution
'''Tips :'''
'''Tips :'''
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# Mix cells before sampling
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# Mix cells before sampling.
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# Take cell sample from top of the liquid
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# Take the cell sample from the top of the liquid.
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# Mix trepan blue into the PBS + cel solution slowly and well before loading
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# Mix Trypan Blue in the PBS + cell solution slowly before loading.

Latest revision as of 00:31, 18 September 2012

Protocol: Trypan Blue Method

Sampling

Cell Density (10^6/ml) Dilution PBS (µl) Cells (µl) Trypan Blue (µl)
1 - 2 4 100 50 50
2 - 4.5 8 125 25 50
4.5 - 7 12 120 15 45
> 7 16 137.5 12.5 50


  1. Take x µL of PBS in a 96-well plate.
  2. Add the required volume of cell culture. Mix once.
  3. Bring the plate back to the microscope, add Trypan Blue to the PBS + cell mixture just before counting the sample.

Trypan Blue is toxic to cells. If left too long with it, even healthy cells will die.


Calculation of LCD :

LCD = Cell Count/(100*4)*Dilution

Tips :

  1. Mix cells before sampling.
  2. Take the cell sample from the top of the liquid.
  3. Mix Trypan Blue in the PBS + cell solution slowly before loading.


Wiki Links

Important pages

Pages

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