MEFs can be grown in vitro on Tissue Culture (TC) treated flasks with growth media based on Dulbecco's Modified Eagle Medium and Fetal Bovine Serum. Growth media is supplemented with other factors, and freeze-down media is supplemented with DMSO. TC treated flasks have more hydrophilic properties than non-TC treated flasks and are important when growing adherence cell lines. MEFs are adherence cells.

All reagents used to create MEF growth media should not be open outside a BSL-2 certified hood. Reserve a time for these hoods and follow all Aseptic Techniques outlined here on the wiki in previous protocols. All contamination in cell culture happens because of improper technique.

This protocol outlines how to make MEF growth media in 50 ml aliquots. Aliquots of each of the materials below should be made in advance of this protocol to streamline creation for your future self and avoid mass-contamination.

• 500 ml Dulbecco's Modified Eagle Medium (DMEM)
• 50 ml Fetal Bovine Serum (FBS)
• Sodium Pyruvate
• B-MeEtOH
• Non-essential Amino Acids (NEAAs)
• Penicillin-streptomycin (P/S)
• L-glutamine (L-G)

• 10 50ml conical tubes
• Automatic pipettor and pipettor tips
• 70% ethanol (EtOH)
• Repeat pipettor and tips with 25/50ml adapters (for sanity)

1. Thaw frozen contents in the heated water bath. (P/S, L-G, FBS)
2. Collect contents in the fridge, spray with 70% EtOH and place upright in the hood. (DMEM, B-MeEtOH, NEAAs, Sodium Pyruvate)
3. Collect 50ml conical tubes and place in hood.
4. Invert DMEM 2-3 times. Pipette 44ml DMEM into each conical tube.
5. Invert sodium pyruvate 2-3 times. Pipette 0.5ml sodium pyruvate into each conical tube.
6. Invert MeEtOH 2-3 times. Pipette 0.0455ml MeEtOH into each conical tube.
7. Invert NEAAs 2-3 times. Pipette 0.05ml NEAAs into each conical tube.
8. Get contents from the water bath, spray with 70% EtOH, place in hood.
9. Shake L-G to ensure full solubility. Pipette 0.5ml L-G to each conical tube.
10. Invert P/S 2-3 times. Pipette 0.05ml P/S into each conical tube.
11. Invert FBS 2-3 times. Remove parafilm. Spray again if parafilm might have been compromised. Pipette 5ml FBS into each conical tube.
12. Label each conical tube with “MEF growth media: DMEM supplemented with… [List all contents added and their amounts]”), parafilm the lid, and store upright in 4*C freezer.

Notes One bottle of DMEM makes 11-12 50ml aliquots of MEF media. To not waste reagents, more supplements can be thawed out and more bottles can be made.

Coming soon…