User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/26: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Last Groninger protocols, splitting cells</span> | ||
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==Materials & Methods== | ==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<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=== | ||
* Defrost FBS @ 4 °C (over the weekend) | |||
* Incubate <u>exactly</u> 30 min. @ 56 °C and put to ice | |||
* Prepare sterile aliquots | |||
* | * Store @ -20 °C | ||
* | |||
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Revision as of 05:48, 7 April 2010
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Summary
Materials & MethodsPlating cells from -80 °C
Putting cells to -80 °C
Preparation of Heat inactivated FBS/FCS
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