User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/26: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(3 intermediate revisions by the same user not shown)
Line 5: Line 5:
{|{{table}} width="800"
{|{{table}} width="800"
|-
|-
|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;">Last Groninger protocols, splitting cells</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>}}
|-
|-
Line 19: Line 19:
===Plating cells from -80 °C===
===Plating cells from -80 °C===
*Take a Ø10cm dish.
*Take a Ø10cm dish.
*Put 10 ml of medium in a 15 ml Falcon tube.
*Put 10 mL of medium in a 15 mL Falcon tube.
*Get the cells out of the -80°C.
*Get the cells out of the -80°C.
*Defrost the cells quikly by using a water bath.
*Defrost the cells quickly by using a water bath.
*As soon as the cells are defrosted, pipet the cells into the medium which is in the Falcon tube.
*As soon as the cells are defrosted, pipette the cells into the medium which is in the Falcon tube.
*Put the medium containing the cells into the petridish.
*Put the medium containing the cells into the petridish.
*Put O.N. at 37°C
*Put O.N. at 37°C
Line 29: Line 29:
*Incubate in fresh medium.
*Incubate in fresh medium.
*If the plate is now confluent the cells can be split and used for experiments.
*If the plate is now confluent the cells can be split and used for experiments.
===Putting cells to -80 °C===
* PREPARE
** Defrost Trypsine (5x diluted)
** 10 mL DMEM S<sup>+</sup> per Ø10cm dish
** 10% DMSO/90% (heat inactivated) FBS solution
* When cells grown in a Ø10cm dish are confluent
* Wash twice with PBS
* Add 1 mL diluted trypsine
* Collect cells in 10 mL DMEM S<sup>+</sup> per Ø10cm dish
* Spin down cells, 1000 RPM, 5 min. RT
* Remove supernatant
* Resuspend pellet in 1 mL 1 10% DMSO/90% FBS solution<sup>*</sup>
* Put 500 μL in a cryovial
* Put vials in a cooling block in the -80 °C freezer
* Next day put them in liquid nitrogen
<br>
<sup>*</sup> Cells don't like DMSO so speed is of essence


===Preparation of Heat inactivated FBS/FCS===
===Preparation of Heat inactivated FBS/FCS===
* Defrost FBS @ 4 °C (over the weekend)
* Defrost FBS @ 4 °C (over the weekend)
* Incubate <u>exactly</u> @ 56 °C and put to ice
* Incubate <u>exactly</u> 30 min. @ 56 °C and put to ice
* Prepare sterile aliquots
* Prepare sterile aliquots
 
* Store @ -20 °C
==Notes==
==Results==
*{{todo| WHAT?}}
 
==Discussion==
*{{done}} Ow..
 
==Related==
===Related entries===
===Same actions===
===Related topics===
==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:FILE.xls| Excel TITLE]]
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
|}
|}


__NOTOC__
__NOTOC__

Revision as of 05:48, 7 April 2010

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

Last Groninger protocols, splitting 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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Plate cells for Anouk
  • Plate cells to bring to Berlin
  • PROTOCOL HEAT INACTIVATE FBS/FCS
  • PROTOCOL USING CELLS FROM -80°C

Materials & Methods

Plating cells from -80 °C

  • Take a Ø10cm dish.
  • Put 10 mL of medium in a 15 mL Falcon tube.
  • Get the cells out of the -80°C.
  • Defrost the cells quickly by using a water bath.
  • As soon as the cells are defrosted, pipette the cells into the medium which is in the Falcon tube.
  • Put the medium containing the cells into the petridish.
  • Put O.N. at 37°C
  • Look the next day if the cells are attached.
  • Wash the cells twice with PBS.
  • Incubate in fresh medium.
  • If the plate is now confluent the cells can be split and used for experiments.

Putting cells to -80 °C

  • PREPARE
    • Defrost Trypsine (5x diluted)
    • 10 mL DMEM S+ per Ø10cm dish
    • 10% DMSO/90% (heat inactivated) FBS solution
  • When cells grown in a Ø10cm dish are confluent
  • Wash twice with PBS
  • Add 1 mL diluted trypsine
  • Collect cells in 10 mL DMEM S+ per Ø10cm dish
  • Spin down cells, 1000 RPM, 5 min. RT
  • Remove supernatant
  • Resuspend pellet in 1 mL 1 10% DMSO/90% FBS solution*
  • Put 500 μL in a cryovial
  • Put vials in a cooling block in the -80 °C freezer
  • Next day put them in liquid nitrogen


* Cells don't like DMSO so speed is of essence

Preparation of Heat inactivated FBS/FCS

  • Defrost FBS @ 4 °C (over the weekend)
  • Incubate exactly 30 min. @ 56 °C and put to ice
  • Prepare sterile aliquots
  • Store @ -20 °C


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/26&oldid=405078"