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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Splitting ASM cells
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |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

 * Dulbecco's Modified Eagle Medium (DMEM) S+(500 mL)
 * Petri dishes
 * Trypsin
 * PBS

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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