User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/04/07: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Preparation first Pilot Berlin</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Materials & Methods==
==Materials & Methods==
===Materials===
===Materials===
*  
* Warm DMEM S<sup>0</sup>
* Warm [http://www.irvinesci.com/techinfo/docs/PBS_Formulation.pdf Dulbecco’s phosphate buffered saline (PBS)]]


===Method===
===Method===
{| border="1"
====Induction 10cm dishes: cAMP precipitation and RII overlay====
|+'''6-wells'''
* Prepare CSE 100% (25 mL) (DMEM S<sup>0</sup>)
!#
* Prepare CSE 15% (6 mL 100% in 40 mL with DMEM S<sup>0</sup>)
!<u>1</u>
* Wash twice the (3) 10cm dishes with PBS
!<u>2</u>
* Add 10 mL of CSE 15% to 2 of 3 plates and S<sup>0</sup> to the third
!<u>3</u>
* Incubate 24 h @ 37 °C
|-
====Prepare cells from -80 °C====
!A
* Cells were plated out from the -80 °C freezer as described [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/26#Preparation_of_Heat_inactivated_FBS.2FFCS|23March2010]]
|
** D9 P21 (prepped 22Jan2010)
|
** D12 P26
|
====Prepare DMEM S<sup>+</sup>====
|-
* FCS was heat inactivated as described [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/26#Preparation_of_Heat_inactivated_FBS.2FFCS|23March2010]]
!B
* All 50 mL was put into DMEM, together with 10 mL PenStrep
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==Notes==
==Notes==
==Results==
* The first CSE apparatus did not function very well and a new one was build using a different peristaltic pump
*{{todo| WHAT?}}
* Although it appeared better than the previous, also this CSE leaves some doubt about the quality where the media did not have smoke coming from it. Beside a fainter than usual smoke scent the media smelled more metallic than before in Groningen.
 
** The difference might also lie within the commercial DMEM from Groningen and the home made DMEM in Berlin
==Discussion==
* The same 25 mL of DMEM S<sup>0</sup> was used to create CSE 100% twice
*{{done}} Ow..
* Stimulation occured at 12:00 hour
 
* All old cultures still left in incubator were thrown out, the recently split cells will be kept untill the new cells are proven to grow sufficiently
* Both D9 and D12 were tested negative for <i>Mycoplasma</i>
* Philipp said that they usually use 5 mL of PenStrep, concentration unknown.. 10 mL will be kept until concentration is known, also due to lack of antifungal
==Related==
==Related==
===Related entries===
===Related entries===
===Same actions===
===Same actions===
===Related topics===
===Related topics===
==Attachment==
<html>
<a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_ht31_160310.xls">
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</a>
</html> [[Media:FILE.xls| Excel TITLE]]
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__NOTOC__
__NOTOC__

Revision as of 05:41, 7 April 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

Preparation first Pilot Berlin <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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Stimulated cells with CSE 15% @ 12 o'clock

Materials & Methods

Materials

Method

Induction 10cm dishes: cAMP precipitation and RII overlay

  • Prepare CSE 100% (25 mL) (DMEM S0)
  • Prepare CSE 15% (6 mL 100% in 40 mL with DMEM S0)
  • Wash twice the (3) 10cm dishes with PBS
  • Add 10 mL of CSE 15% to 2 of 3 plates and S0 to the third
  • Incubate 24 h @ 37 °C

Prepare cells from -80 °C

  • Cells were plated out from the -80 °C freezer as described 23March2010
    • D9 P21 (prepped 22Jan2010)
    • D12 P26

Prepare DMEM S+

  • FCS was heat inactivated as described 23March2010
  • All 50 mL was put into DMEM, together with 10 mL PenStrep

Notes

  • The first CSE apparatus did not function very well and a new one was build using a different peristaltic pump
  • Although it appeared better than the previous, also this CSE leaves some doubt about the quality where the media did not have smoke coming from it. Beside a fainter than usual smoke scent the media smelled more metallic than before in Groningen.
    • The difference might also lie within the commercial DMEM from Groningen and the home made DMEM in Berlin
  • The same 25 mL of DMEM S0 was used to create CSE 100% twice
  • Stimulation occured at 12:00 hour
  • All old cultures still left in incubator were thrown out, the recently split cells will be kept untill the new cells are proven to grow sufficiently
  • Both D9 and D12 were tested negative for Mycoplasma
  • Philipp said that they usually use 5 mL of PenStrep, concentration unknown.. 10 mL will be kept until concentration is known, also due to lack of antifungal

Related

Related entries

Same actions

Related topics


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