jcMEF-ES Culture

jcMEF-ES Culture

Thaw MEFs (murine embryonic fibroblasts)

Remove the vial of MEFs from the liquid nitrogen container or from -80━ C freezer (transfer around on dry ice if needed). Immediately put it into 370C water, move around without leaving the water until it just thawed.

Spray the vial with 70% ethanol and wipe it briefly. Transfer the contents of the vial into a 15ml tube contain 10mL MEF media in a tissue culture hood.

Spin at 1200RPM 5 minutes at room temperature.

Aspirate the media away and add 5mL fresh media, and gently resuspend the cells.

Transfer the resuspend cell in the T-75 flask, add 10mL more fresh media, then culture in 370C 5% CO2 incubator.

Change media the next day. Pass by 1 to 2 when confluent (usually 2 more days later).

Important: Get things ready and set. Carry out as quickly as possible.

Pass the MEFs

Aspirate the media away.

Wash the cell layer with DPBS by adding 10mL DPBS, Lay down the flask to make sure the cell layer is immerge under the DPBS.

Aspirate the DPBS away, and add 2mL 0.05% trypsin-EDTA. Turn the flask so that Trypsin solution covers the flask bottom evenly.

Put the flask in the incubator until the MEFs come loose and come off the bottom.

Add 10mL media, beat the MEFs into single cells by pipeting up and down.

Transfer half to a new same size flask, add 10mL fresh media to each flask. Shake gently to mix even and put all flasks into the CO2 incubator.

Prepare MEFs to Feeder cell fro ES cells

MEFs must be inactivated before being used as feeder cells. MEFs can inactivated either by Gamma irradiation or by mitomycin C solution (10レg/ml in MEF media) treatment. Please contact us for detail procedure. Alternatively, we provide irradiated MEFs for immediately ES feeding need. This is handy when you only do a small scale ES cell culture such as for microinjection.

Inactivated MEFs are the plated on 0.1% gelatin treated surface, grow to confluency to function as feeders.

Inactivate MEFs by irradiation (preferred)

Trypsinized the MEFs, beat them into single cells (see Pass the MEFs). Collect MEFs into 50mL tube, spin and resuspend into DPBS, spin down a second time and resuspend into fresh medium this time.

Irriadiate with gamma ray at dosage of 2000- 3000 rads (usually blood bank or Radiology Department are equipped with gamma irradiators).

Plate the irradiated MEFs in gelatin coated dish or flask. 2x106 for each 100mm dish or T-75 flask.

Culture the inactivated MEFs over night. The MEFs will attach and grow to form a layer on the gelatin, ready to be feeders.



Inactivation of MEFs by mitomycin C

1. Aspirate away media, wash with 5 - 10 ml DPBS. 2. Add 7 ml of the mitomycin C solution (10レg/ml in MEF media) 3. Incubate for 3 hrs. 4. During this time, coat 100 mm TC dish or T-75 flask with 10mL 0.1% gelatin inDPBS. Make sure whole bottom of the dish or flask was cover by the gelatin solution. 5. When 3 hour time is up, aspirate the mitomycin C, wash with 10ml DPBS 3 times 6. Trypsinize the MEF, add media and beat the MEF into single cells. 7. Grow the inactivated MEFs on the gelatin treated dish or flask. 2x106 for each 100mm dish or T-75 flask 8. Culture the inactivated MEFs over night. 9. The MEFs will attach and grow to form a layer on the gelatin, ready to be feeders.

Freeze MEFS MEFs can be frozen for later use no mater they are inactivated or not. Harvest the MEFs, count and adjust density to 2X107cell/mL. Aliquot 0.5mL to each cryotube. Add equal volume 2x freezing medium, and mix by gentle inversion. Put the tubes into ^10C freeze container ̄, put the whole container into -800C freezer to freeze. Move the tubes to liquid nitrogen container for long term storage.

Order Information and Medium formula:

Name Company Catalog number Geneticin GIBCO 11811-031 DMEM GIBCO 11965 L-Glutamine (100X) GIBCO 25030-081 MEM NEAA (100X) GIBCO 11140-050 PEN./STREP. GIBCO 15070-050 Trypsin-EDTA(0.05%) GIBCO 25300-054 DPBS GIBCO 21600-010 2-Mercaptoethanol Sigma M7522-100ML DMSO Sigma 494429-1L Gelatin Type A Sigma G1890 ESGRO LIF Chemicon ESG1107 Cryo 10C Freeze Container Nalgene 5100-0001 Regular Fetal Bovine Serum Hyclone SH30088.03 ES Fetal Bovine Serum Hyclone SH30070.03E

MEF Media (500 mLs): 435mls DMEM 50mls regular Fetal Bovine Serum 5mls L-Glutamine 5mls MEM Non Essential Amino Acids (NEAA) 5mls Penicllin-Streptomycin Sterilized by 0.22レm filter

ES Media (500 mLs): 410mls DMEM 75mls ES cell qualified Fetal Bovine Serum 5mls L-Glutamine 5mls MEM Non Essential Amino Acids (NEAA) 5mls Penicllin-Streptomycin 50レL LIF (1,000 units/mL final concentration) 5レL 2-ME (2-Mercaptoethanol) Sterilized by 0.22レm filter

Freezing Media: 60mls ES Media 20mls Regular FCS 20mls DMSO Sterilized by 0.22レm filter