Based on splitting onto on plate
- Warm collagenase media to 37 °C in a water bath.
- Aspirate media off of cell culture plate.
- Add the following amount of collagenase
- 0.5ml/well of 4 well plate
- 1.0ml /well of 6 well plate
- Incubate at 37 °C for 5-10 minutes; stop incubation when edges of colonies begin to pull away from the plate.
- Aspirate the collagenase and add appropriate volume (3ml) of conditioned media (CM) to the plate (see notes).
- Collect cells off of the plate by scraping and washing with the CM, transfer to a 15ml tube.
- Aspirate Matrigel off of 6 well plate (see Matrigel aliquoting and plating procedure)
- Add 2ml of CM per well of 6 well plate.
- Add 5ul bFGF.
- Plate 0.4ml/well into each well until there is approximately 0.6 ml remaining.
- Add the remaining 0.6ml dropwise to each well until done.
- Make sure the cells are evenly distributed across the plate.
- Place gently into incubator.
- Let settle overnight.
- High density MEFs for CM last about 2 weeks after plating. Be sure to check them regularly for cell death.
- bFGF is stable at 4 °C for one month.