- 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
- 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