User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/16: Difference between revisions
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===Materials=== | ===Materials=== | ||
* hTERT (Airway smooth muscle cells, ASMC's) | * hTERT (Airway smooth muscle cells, ASMC's) | ||
* [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1421&ckt=1 InCELLect™ AKAP St-<b>Ht31</b> Inhibitor Peptide (Promega)] | * [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1421&ckt=1 InCELLect™ AKAP St-<b>Ht31</b> Inhibitor Peptide (Promega)] (10 mM) | ||
* [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1422 InCELLect™ St-<b>Ht31P</b> Control Peptide] | * [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1422 InCELLect™ St-<b>Ht31P</b> Control Peptide] | ||
* <b>8-pcpt</b>-2'-o-me-camp | * <b>8-pcpt</b>-2'-o-me-camp | ||
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<u>Stock solutions</u> | <u>Stock solutions</u> | ||
* HT31 (45 μL HT31 + 3 mL DMEM S<sup>0</sup>) | * HT31 (45 μL HT31 + 3 mL DMEM S<sup>0</sup>) (10 mM) | ||
** 270 μL HT31 | ** 270 μL HT31 | ||
** 18 mL DMEM S<sup>0</sup> | ** 18 mL DMEM S<sup>0</sup> | ||
* HT31P (45 μL HT31 + 3 mL DMEM S<sup>0</sup>) | * HT31P (45 μL HT31 + 3 mL DMEM S<sup>0</sup>) | ||
** 270 μL HT31P | ** 270 μL HT31P | ||
** 18 mL DMEM S<sup>0</sup> | ** 18 mL DMEM S<sup>0</sup> | ||
* 8-pctp | * 8-pctp (60 μL in 2 mL) (10 mM) | ||
** 360 μL | ** 360 μL | ||
** 12 mL DMEM S<sup>0</sup> | |||
* 6-Benz (300 μL in 2 mL) (10 mM) | |||
** 1800 μL | |||
** 12 mL DMEM S<sup>0</sup> | ** 12 mL DMEM S<sup>0</sup> | ||
* CSE 45% | * CSE 45% | ||
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===Method=== | ===Method=== | ||
* Remove medium | * Remove medium from 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 200 μL DMEM | * Add 200 μL DMEM S<sup>0</sup> with either HT31 or HT31P (see schedule below) | ||
** Incubate 20 min. | ** Incubate 20 min. | ||
* Add 400 μL DMEM S<sup>0</sup> | * Add 400 μL DMEM S<sup>0</sup> to CTR (lane A) | ||
* Add 200 μL DMEM S<sup>0</sup> | * Add 200 μL DMEM S<sup>0</sup> to CSE (lane B) | ||
* Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D) | * Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D) | ||
* Wait 25 min. | * Wait 25 min. | ||
** Prepare 25 mL DMEM S<sup>0</sup> | ** Prepare 25 mL DMEM S<sup>0</sup> with 100% CSE | ||
** Dilute 100% CSE to 45% CSE | ** Dilute 100% CSE to 45% CSE | ||
* Add 200 μL 45% CSE to wells according to schedule below (lane B, C D) | * Add 200 μL 45% CSE to wells according to schedule below (lane B, C D) | ||
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|- | |- | ||
|<b>A</b><BR> (CTR) | |<b>A</b><BR> (CTR) | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
|- | |- | ||
|<b>B</b><BR> (CSE) | |<b>B</b><BR> (CSE) | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
|- | |- | ||
|<b>C</b><BR> (CSE+8-pcpt) | |<b>C</b><BR> (CSE+8-pcpt) | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
|- | |- | ||
|<b>D</b><BR> (CSE+6-Benz) | |<b>D</b><BR> (CSE+6-Benz) | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:red"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
| | |style="background:green"| | ||
|- | |- | ||
|} | |} | ||
<b>CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp</b> | <b>CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp</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]] | ||
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|} | |} | ||
__NOTOC__ | __NOTOC__ |
Revision as of 08:22, 16 May 2010
<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>
Stimulation hTERT cells | <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 look at the effect of Protein kinase A (PKA) and Epac on the IL-8 production by airway smooth muscle cells (ASMC's) induced by cigarette smoke extract (CSE), 8-pcpt-2'-o-me-camp (8-pcpt) and 6-Benz-cAMP (6-Benz) are used to activate Epac and PKA respectively. To illustrate the influence of A kinase Anchoring Proteins (AKAP's) an allround inhibitor of PKA (subunit RII) binding to AKAP's HT31 is used. An inactive peptide HT31P is used as an control. The purpose of the described method is to stimulate the cells with the substances to prepare for future experiments (e.g. ELISA, Western blot) Materials & MethodsMaterials
Stock solutions
Method
CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp Related entriesRun 1: Ht31/Ht31P D9/D12 |