User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/18: Difference between revisions
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==Summary== | ==Summary== | ||
To determine the amount of interleukin 8 (IL-8) produced by the airway smooth muscle cells (ASMC's) [http://en.wikipedia.org/wiki/ELISA ELISA] is performed using the PeliKine Compact™ human IL-8 ELISA kit. In this way the effect of PKA, EPAC and CSE on the inflammatory cytokine can be determined. | |||
==Materials & Methods== | ==Materials & Methods== | ||
===Materials=== | ===Materials=== | ||
* Coating buffer | * Coating buffer | ||
* IL-8 antibody | * IL-8 antibody Coating antibody | ||
* 96-wells plate (MaxiSorb) | * 96-wells plate (MaxiSorb) | ||
* [[PBS]] | * [[PBS]] 5% | ||
* [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] | * [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] 5% (in PBS) | ||
* [http://www.sanquin.pl/datasheet/m1918.pdf Pelikine Compact™ Human IL-8 ELISA kit] | * [http://www.sanquin.pl/datasheet/m1918.pdf Pelikine Compact™ Human IL-8 ELISA kit] | ||
* Washing buffer | * Washing buffer (PBS + 0.005% TWEEN 20) | ||
* IL-8 antibody | * IL-8 antibody | ||
* Streptavidin conjugated to HRP | |||
* Substrate buffer | |||
** 1.5 sodiumacetate, resuspend in 80 mL ultrapure water | |||
** pH 5.5 | |||
** up to 100 mL with ultrapure water | |||
* TMB stock solution (light sensitive) | |||
** 30 mg [http://en.wikipedia.org/wiki/3,3%E2%80%99,5,5%E2%80%99-Tetramethylbenzidine TMB] | |||
** 5 mL [http://en.wikipedia.org/wiki/Dimethyl_sulfoxide DMSO] | |||
===Method=== | ===Method=== | ||
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|100/100 | |style="background:red"|100/100 | ||
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|100/100 | |style="background:red"|100/100 | ||
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|B | |B | ||
|150/50 | |style="background:green"|150/50 | ||
|150/50 | |style="background:green"|150/50 | ||
|150/50 | |style="background:green"|150/50 | ||
|175/25 | |style="background:blue; color:white" |175/25 | ||
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|D | |D | ||
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* Add 100 μL of diluted sample | * Add 100 μL of diluted sample | ||
* Incubate ≥1h @ RT | * Incubate ≥1h @ RT | ||
* | * Wash 5x 200 μL Washing buffer | ||
* Remove buffer | * Remove buffer | ||
** {{todo| prepare biotinylated antibody}} | ** {{todo| prepare biotinylated antibody}} | ||
* Add 100 μL Biotinylated antibody | * Add 100 μL Biotinylated antibody | ||
* Incubate 1h @ RT | * Incubate 1h @ RT | ||
* Remove antibody | |||
* Wash 5x 200 μL Washing buffer | |||
* Add 100 μL streptavidin conjugated to HRP | |||
* Incubate 25 min. @ RT | |||
* Remove liquid | |||
* Wash 5x 200 μL Washing buffer | |||
** {{todo| prepare TBM substrate}} | |||
** 1.2 μL H<sub>2</sub>O<sub>2</sub> (30%) | |||
** 400 μL TBM stock solution | |||
** 24 mL substrate buffer | |||
* Add 100 μL substrate | |||
* Incubate 30 min. @ RT | |||
* Stop reaction, add 100 μL stopping solution | |||
** It will turn yellow | |||
* Measure absorbance @ 450 nm | |||
** Check duplo standard curve | |||
** Check if values fall within standard curve (preferred) | |||
<!--{| border="1" | <!--{| border="1" | ||
|+'''96-wells plate''' | |+'''96-wells plate''' | ||
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* Of the 96-wells plate well F1 contain a Polystyrene fragment | * Of the 96-wells plate well F1 contain a Polystyrene fragment | ||
==Results== | ==Results== | ||
* | * Standard curves were accurate (R square 1>0.99) | ||
* Negative controls showed hardly any IL-8 secretion into the medium (see figures below) | |||
[[Image:AverageIL8secretionD918022010.JPG|500px]]<BR> | |||
<b>Image 180220101: The average IL-8 secretion of donor 9 corrected for [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17| cell viability]]. Error bars represent standard deviation. CTR: control, CSE: Cigarette smoke extract, 8-p: 8-pcpt-2'-o-me-camp, Bnz:6-Benz-cAMP.</b><BR> | |||
[[Image:AverageIL8secretionD1218022010.JPG|500px]]<BR> | |||
<b>Image 180220102: The average IL-8 secretion of donor 12 corrected for [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17| cell viability]]. Error bars represent standard deviation. CTR: control, CSE: Cigarette smoke extract, 8-p: 8-pcpt-2'-o-me-camp, Bnz:6-Benz-cAMP.</b><BR> | |||
* Donor 9 showed twice as low IL-8 concentrations (see figures | |||
==Conclusion== | ==Conclusion== | ||
* | *It appears that HT31P affects the cells viability negatively. | ||
*When comparing the effect of CSE without (HT31P) and with (HT31) PKA/AKAP interaction inhibition, it seems that when AKAP/PKA association is inhibited it decreases CSE induced IL-8 release. | |||
* | ** PKA/AKAP interaction is required for CSE induced IL-8 secretion. | ||
* | * When inducing Epac no effect is seen when comparing the HT31 situation, however slight decrease of IL-8 production is seen when HT31P control situation receives Epac stimulation. | ||
* | ** When Epac can compete with PKA for the present cAMP it inhibits the increased IL-8 secretion. | ||
* | ** Epac does not have an influence when PKA can not have its effect | ||
* | * Increased PKA activity induces inhibition of IL-8 secretion even without AKAP association | ||
** It might be that PKA is activated prior to AKAP association, removing its necessity | |||
*<b>PKA/AKAP induces IL-8 secretion, however an excess of active PKA with/without AKAP association lowers IL-8 secretion</b> | |||
** Ht31 does not prevent the activation of PKA, only its localization close to cAMP sources and PKA effectors | |||
*<b> PKA is the more important effector of cAMP compared to Epac</b> | |||
==Related entries== | |||
===Run 1: Ht31/Ht31P D9/D12=== | |||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/10|Splitting cells 10Feb2010]] | |||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/12|Counting cells 12Feb2010]] | |||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/15|Putting to S<sup>0</sup> 15Feb2010]] | |||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16|Stimulating cells 16Feb2010]] | |||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17|Ending stimulation & Viability 17Feb2010]] | |||
==Attachment== | |||
<html> | |||
<a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_ht31_160310.xls"> | |||
<img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> | |||
</a> | |||
</html> [[Media:HTERT_Donor_9_ht31_180210.xls| Excel hTERT D9]] | |||
<br> | |||
<html> | |||
<a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_ht31_160310.xls"> | |||
<img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> | |||
</a> | |||
</html> [[Media:HTERT_Donor_12_ht31_180210.xls| Excel hTERT D12]] | |||
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Revision as of 01:19, 26 March 2010
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ELISA | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||
SummaryTo determine the amount of interleukin 8 (IL-8) produced by the airway smooth muscle cells (ASMC's) ELISA is performed using the PeliKine Compact™ human IL-8 ELISA kit. In this way the effect of PKA, EPAC and CSE on the inflammatory cytokine can be determined. Materials & MethodsMaterials
MethodCoating of plate
Preparing Sandwich
Notes
Results
Conclusion
Related entriesRun 1: Ht31/Ht31P D9/D12
Attachment<html> <a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_ht31_160310.xls"> <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> </a>
</html> Excel hTERT D9
<img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> </a> </html> Excel hTERT D12 |