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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Last Groninger protocols, splitting cells
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

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

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


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