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Initial culture conditions

Matings are set-up on Sunday and plugs are checked on Monday for delivery of mice on Thursday. On the Wednesday prior to dissecting embryos, prepare 4-well plates with PMEFs as follows:

Establishing monolayers of PMEFs

Components:

• Iscove's modified Dulbecco's medium (IMDM); Gibco/Invitrogen #12440-053 for 500ml; 12440-046 for 1 L.
• Hyclone FBS tested for ES cells; heat inactivated; 40ml aliquots; stored -20. Characterized Fetal Bovine Serum, Catalog #SH30071, Lot#ARH27145.
• 100x Pen/strep: Gibco/Invitrogen #15070-063 for 100ml; 10ml aliquots; store -20.
• Primary mouse embryonic fibroblast feeder layers (PMEFs), Mitomycin-C treated. Purchased from Specialty Media (now Millipore), PMEF-CF.
• Gelatinized tissue culture dishes:
• .1% gelatin (typeB from bovine skin, Sigma G9382); .3g in 300ml ddH2O autoclaved; cover dishes with solution and aspirate off; allow to air dry in hood--approximately 1/2 hour (alternatively can allow to air dry overnight): use within 24 hours.
• 4-well plates: Nunc multidish 4wells (176740).

PMEF-medium/50 ml:

• 44.5ml Iscove's Modified Dulbecco's Medium
• 5ml FBS (10% final)
• 0.5ml 100x PEN/STREP (50 u or µg/ml final)

(1) Rapidly thaw 1 vial of PMEF-CF cells in a waterbath and transfer to 10ml of PMEF-medium. Spin down and resuspend into 25ml PMEF-medium.

(2) Transfer 0.5ml/well onto several gelatinized 4-well plates. {Depending on the expected yield of embryos, you will want 6-10 plates total}

(3) Incubate overnight. Verify the presence of a mono-layer prior to use. {Mono-layers are good for about 2 weeks}

Preparing PMEF plates for embryo culture

Prior to culturing embryos, replace PMEF-medium with SR-medium.

Recipe for SR-medium

Components:

• Iscove's modified dulbecco's medium (IMDM); Gibco/Invitrogen #12440-053 for 500ml; 12440-046 for 1 L; store 4 degrees and protect from light.
• Knockout SR serum replacement for ES cells (SR); Gibco 10828-028; 10ml aliquots; stored -20. DO NOT HEAT INACTIVATE.
• 100x 2-mercaptoethanol (Sigma M7522, 14.3 M stock): dilute 70 microliters into 100ml of sterile ddH2O and filter sterilize; 10ml aliquots; store 4 degrees.
• 100x MEM nonessential amino acids: Gibco/Invitrogen #11140-050 for 100ml; 10ml aliquots; store 4 degrees.
• 100x Pen/strep: Gibco/Invitrogen #15070-063 for 100ml; 10ml aliquots; store -20.
• Recombinant LIF protein, produced as per the V. Prideaux protocol; filter sterilized; put into 30 microliter aliquots; flash frozen in liquid nitrogen and stored -80; each batch tested for efficacy against older batches.
• Alternatively LIF can be purchased from Chemicon (ESGRO 10⁷ units: ESG1107; enough for 10L of medium) and supplemented at 10³units/ml.

SR-medium/50 ml:

• 40ml Iscove's MDM (contains 4 mM L-glutamine and 1 mM sodium pyruvate)
• To an aliquot of IMDM add in exactly the order listed:
• 10ml SR (20% final)
• 0.5ml 100x (10 mM) 2-mercaptoethanol (.1mM final)
• 0.5ml 100x MEM nonessential amino acids (.1 mM final)
• 0.5ml 100x PEN/STREP (50 u or µg/ml final)
• 30 µl of LIF (one -80 aliquot)

(4) Aspirate PMEF-medium from 4-well plates and replace with 1.0ml SR-medium. Return to incubator and allow to equilibrate for several hours prior to culturing embryos. {Do this prior to fetching your uteri}

 

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