User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/04/07

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Preparation first Pilot Berlin Main project page
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Summary

  • Stimulated cells with CSE 15% @ 12 o'clock

Materials & Methods

Materials

Method

Induction 10cm dishes: cAMP precipitation and RII overlay

  • Prepare CSE 100% (25 mL) (DMEM S0)
  • Prepare CSE 15% (6 mL 100% in 40 mL with DMEM S0)
  • Wash twice the (3) 10cm dishes with PBS
  • Add 10 mL of CSE 15% to 2 of 3 plates and S0 to the third
  • Incubate 24 h @ 37 °C

Prepare cells from -80 °C

  • Cells were plated out from the -80 °C freezer as described 23March2010
    • D9 P21 (prepped 22Jan2010)
    • D12 P26

Prepare DMEM S+

  • FCS was heat inactivated as described 23March2010
  • All 50 mL was put into DMEM, together with 10 mL PenStrep

Notes

  • The first CSE apparatus did not function very well and a new one was build using a different peristaltic pump
  • Although it appeared better than the previous, also this CSE leaves some doubt about the quality where the media did not have smoke coming from it. Beside a fainter than usual smoke scent the media smelled more metallic than before in Groningen.
    • The difference might also lie within the commercial DMEM from Groningen and the home made DMEM in Berlin
  • The same 25 mL of DMEM S0 was used to create CSE 100% twice
  • Stimulation occured at 12:00 hour
  • All old cultures still left in incubator were thrown out, the recently split cells will be kept untill the new cells are proven to grow sufficiently
  • Both D9 and D12 were tested negative for Mycoplasma
  • Philipp said that they usually use 5 mL of PenStrep, concentration unknown.. 10 mL will be kept until concentration is known, also due to lack of antifungal

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