EmbryoMax^® Primary Mouse Embryonic Fibroblasts

Plating MEF Feeder Cells

Procedure:

1. Prior to thawing PMEF feeder cells, coat plates/flasks with Gelatin solution.
2. Thaw PMEF vial(s) quickly in a 37 °C water bath and transfer to a 15 mL tube (already containing 10 mL of warm PMEF Feeder Cell Medium). Gently invert the tube to distribute, and centrifuge at 300 xg for 4–5 minutes.
3. Remove supernatant and resuspend the cell pellet in warm PMEF Feeder Cell Medium.
4. Remove the Gelatin solution from plates/flasks, and aliquot the PMEF feeder cell suspension at the densities recommended in Table 4.1 of the mouse ES protocol guide
5. Incubate the PMEF Feeder cells at 37 °C with 5% CO2. Use Figures 4A, B and C in the mouse ES protocol guide as a guide for an estimate of correct PMEF density and
appearance. Gelatinized plates may be used for 12–14 days.

The EmbryoMax range of PMEF cells provides researchers with a convenient solution for ES cell culture by eliminating the need for time consuming feeder cell isolation and preparation. Many embryonic stem cell culture protocols necessitate the use of primary mouse embryo fibroblast (PMEF) cells. In these protocols, ES cells are typically cultured on a monolayer of PMEF feeder cells. Feeder cells perform two important roles in stem cell culture: they secrete several important growth factors into the medium, which help maintain pluripotency, and they provide a cellular matrix for ES cells to grow.

The DR4 strain of MEF feeder cells are resistant to neomycin, hygromycin, puromycin, and 6-thioguanine. They are mitotically arrested by mytomycin-C treatment and will not proliferate.

Primary Mouse Embryo Fibroblasts

EmbryoMax PMEF, P3, Strain DR4, Mito-C Treated: (Part number PMEF-DR4) One (1) vial containing ≥4 x10^6 cells per vial.

Cell Viability and Morphology: PASSED
Mycoplasma Testing: PASSED
Bacterial Testing: PASSED
Fungi Testing: PASSED

Vials should be stored in the vapor phase of liquid nitrogen.

EmbryoMax is a registered trademark of Merck KGaA, Darmstadt, Germany