8/17/2019 Protocolo de produção de MEFs (mouse embryonic fibroblasts)

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MEF (mouse embryonic fibroblasts) Protocol

1. Set up timed pregnancies, fetuses should be harvested between day 12.5-14.5d of gestation.

2. Remove the entire uterus and place it in a 5m! tube with "#S.

$. %nder the hood, cut the uterus in pieces, each containing one gestational sac."lace in & well plates.

4. Remove mother tissues and 're-plate( the embryos in new & well plates with"#S.

5. )arefully remove the embryo head and red organs. %se a piece of the headfor genotyping.

&. Remove all blood from embryo and wash with 1-2ml of "#S, put tissue into anew & well plate.

*. +ince the tissue with a blade and add $ ml of trypsin ,5 solution.

/. 0ncubate at $*°) for 1 min. shaing 2-$. 3dd more 2ml of trypsin solution

and pipet up and down several times. 0ncubate more some minutes todissociate the tissue entirely.

. 3dd 1ml of #S plus 1ml +6 media to neutrali7e trypsin and transfer to5ml tube to spin down at $5g /min.

1.)arefully aspirate the supernatant with 1ml pipet leaving $ to 5 ml and thepellet. Re-suspend in 1ml +6 media and put in a 1cm plate.

11. Replace the media ne8t day. ree7e down " or split 19$ when culturesbecome confluent.

+aterials

:wo forceps

;ne scissor

:rypsin solution .5