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

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Last Groninger protocols, splitting cells 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

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


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