User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/16: Difference between revisions
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* Supernatants collected [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/05|5March2010]] (D12), [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/10|10March2010]] (D12, P25) & [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/11|11March2010]] (D9, P21) | * Supernatants collected [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/05|5March2010]] (D12), [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/10|10March2010]] (D12, P25) & [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/11|11March2010]] (D9, P21) | ||
* [[PBS]] 5% | * [[PBS]] 5% | ||
* [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] 5% in PBS | * [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] 5% in PBS (20 mL per 96-wells) | ||
* [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 (PBS + 0.005% TWEEN 20) | * Washing buffer (PBS + 0.005% TWEEN 20) | ||
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** 30 mg [http://en.wikipedia.org/wiki/3,3%E2%80%99,5,5%E2%80%99-Tetramethylbenzidine TMB] | ** 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] | ** 5 mL [http://en.wikipedia.org/wiki/Dimethyl_sulfoxide DMSO] | ||
* Dilution buffer (1:5, 32 mL H<sub>2</sub>O, 8 mL undiluted buffer) | |||
===Method=== | ===Method=== | ||
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* Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S<sup>0</sup>) | * Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S<sup>0</sup>) | ||
* Rinse twice with warm PBS and remove buffer | * Rinse twice with warm PBS and remove buffer | ||
* Add | * Add 300 μL DMEM S<sup>0</sup> with HT31P 0 - 50 μM (see schedule below) | ||
** Incubate 20 min. | ** Incubate 20 min. | ||
** Prepare 25 mL DMEM S<sup>0</sup> with 100% CSE | ** Prepare 25 mL DMEM S<sup>0</sup> with 100% CSE | ||
** Dilute 100% CSE to | ** Dilute 100% CSE to 30% CSE (3 mL CSE 100% + 7 mL S<sup>0</sup>) | ||
* Add | * Add 300 μL 30% CSE to wells according to schedule below (lane C & D) | ||
* Incubate 24 h. | * Incubate 24 h. | ||
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|} | |} | ||
<b>CTR = control, CSE = Cigarette Smoke extract</b> | <b>CTR = control, CSE = Cigarette Smoke extract</b> | ||
====[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/18|ELISA]]==== | ====[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/18|ELISA]]==== | ||
<b><u>Preparing Sandwich</u></b> | <b><u>Preparing Sandwich</u></b> | ||
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* Stop reaction, add 100 μL stopping solution | * Stop reaction, add 100 μL stopping solution | ||
** It will turn yellow | ** It will turn yellow | ||
* Measure absorbance @ | * Measure absorbance @ 450 nm | ||
** Check duplo standard curve | ** Check duplo standard curve | ||
** Check if values fall within standard curve (preferred) | ** Check if values fall within standard curve (preferred) | ||
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|- | |- | ||
|} | |} | ||
====Co-immunoprecipitation==== | ====Co-immunoprecipitation==== | ||
* resuspend beads prepared [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/15| 15March2010]] | * resuspend beads prepared [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/15| 15March2010]] | ||
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* Incubate ON @ °C | * Incubate ON @ °C | ||
====Splitting cells==== | |||
* D9 P23 & D12 P27 were split, one petridish to 4 (P+1) | |||
==Results== | ==Results== | ||
{| border="1" | {| border="1" | ||
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==Conclusion== | ==Conclusion== | ||
*{{done}} Ow.. | *{{done}} Ow.. | ||
==Related== | ==Related entries== | ||
* [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| Splitting cells]] | ====Run 4: Ht31P dose/response curve D9/D12==== | ||
* [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/15| Cells put to S<sup>0</sup>]] | * [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| Splitting cells 12March2010]] | ||
* [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/15| Cells put to S<sup>0</sup> 15March2010]] | |||
====Run 3: Ht31/S<sup>0</sup>==== | |||
*Counting cells | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/26|26Feb2010 D9/D12]] | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/05|03March2010 D9 (P21)/D12 (P25)]] | |||
*Putting to S<sup>0</sup> | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/03|03March D9/D12]] | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/08|08March2010 D12 (P25)]] | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/09|09March2010 D9 (P21)]] | |||
*Stimulating cells | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/04|04March2010 D9 (samples lost)/D12]] | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/09|09March2010 D12 (P25)]] | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/10|10March2010 D9 (P21)]] | |||
*Ending stimulation & Viability | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/05|05March2010 D12]] | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/10|10March2010 D12 (P25)]] | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/11|11March2010 D9 (P21)]] | |||
*ELISA | |||
**[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/16|16March2010 D12, D12 (P25) & D9 (P21)]] | |||
==Same actions== | ==Same actions== | ||
* [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Cell stimulation]] | * [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Cell stimulation]] | ||
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</a> | </a> | ||
</html> [ | </html> [[Media:HTERT_Donor_12_ht31_160310.xls| Excel hTERT D12]] | ||
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</html> [ | </html> [[Media:HTERT_donor_12_P25_160310.xls| Excel hTERT D12 P25]] | ||
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</a> | </a> | ||
</html> [ | </html> [[Media:HTERT_Donor_9_P21_160310.xls| Excel hTERT D9 P21]] | ||
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Revision as of 14:38, 18 March 2010
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ELISA & hTERT stimulation | <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> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Summary
Materials & MethodsMaterialsStimulation
ELISA
MethodStimulation hTERT cells
CTR = control, CSE = Cigarette Smoke extract ELISAPreparing Sandwich
Co-immunoprecipitation
(possible also beads (30 μL) with only sample (200 μL))
Splitting cells
Results
Conclusion
Related entriesRun 4: Ht31P dose/response curve D9/D12Run 3: Ht31/S0
Same actionsAttachment<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 D12 <html> <a href="http://openwetware.org/wiki/Image:HTERT_donor_12_P25_160310.xls"> <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> </a> </html> Excel hTERT D12 P25 <html> <a href="http://openwetware.org/wiki/Image:HTERT_Donor_9_P21_160310.xls"> <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> </a> </html> Excel hTERT D9 P21 |