MEF Production Protocol

1) Set up appropriate mice together

2) At 13.5 days after plugging, remove uterus from pregnant mice, hold in filtered PBS

3) Remove individual embryos, remove head for genotyping, remove placenta, umbilical chord and internal organs (heart, liver, kidney & gut) from mouse embryo.

4) Wash in PBS

5) Using a scalpel blade mince embryo until your arm hurts.

6) To mouse add 1ml trypsin EDTA. Then mince it some more. Use a filtered 1 ml tip to break down tissue.

7) Incubate mouse/trypsin mixture for 30 mins at 37°C, aspirating at 10 min intervals.

8) Wash out trypsin using 6ml of the following pre-warmed media. Centrifuge at 1000rpm for 5 mins. A jelly-like substance may form in the supernatant – keep this, discard the rest of supernatant.

GIBCO 1196010% FBS5ml L-glutamine x 1005ml Pen-Strep x 100

9) Plate out cells, incubate with 10 ml media. 10) Incubate 1-2 days replate all cells and grow to confluence. Trypsinise cells

and freeze in 10% DMSO mark passage 0.