User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/10

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Splitting ASM cells Main project page
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Summary

To keep cells viable they need to be divided (split) over multiple plates after they have become confluent. This provides cells with space to keep dividing.

Materials & Methods

Materials

Method

  • Warm solution required in water (PBS, DMEM S+)
  • Switch on laminar flow, clean it and spray everything required (except for cells) with EtOH (70%) prior to putting into the laminar flow
  • Switch the vacuum pump on
  • Check cells under microscope
  • Remove medium with vacuum pump
  • Rinse cells twice with 5 mL PBS and remove buffer with vacuum pump
  • Defrost trypsin
  • Add 1 mL trypsin
  • Incubate 37 °C, 5 min.
    • Prepare Greiner tube with 39 mL DMEM S+
    • Prepare 3 Petri dishes with Name, Donor and Passage
  • After incubation add 5 - 10 mL DMEM S+ to cells (from prepared 39 mL)
    • Rinse the dish with the medium in order to get all cells removed from plate and in the liquid
  • Remove cell suspension and add to the remaining medium in the prepared Greiner tube (39 mL + 1 mL trypsin = 40 mL total volume)
  • Divide cells over the 4 petri dishes (recycle previous one) 10 mL each
  • Incubate @ 37 °C


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