DAY 1
Take Neo resistant embryos at E12.5-E13.5.
Evicerate and remove embryo heads in cold, sterile PBS.
Wash embryos 3x in sterile PBS.
Combine one litter of embryos in corner of a petri dish, add ~3 drops warm trypsin.
Chop tissue with scissors continuously for 5 minutes in TC hood.
Incubate at 37°C for 10 min. (Prop up plate on one side so tissue is concentrated to one side of dish)
Triturate and transfer cells + ~25 ml warm media to 50 ml conical.
Let large pieces settle to bottom, remove sup to new tube.
Triturate large tissue pieces, let settle, and collect supernatant 2 more times with ~5ml media each.
Aliquot cells so that there is 1 embryo/10 cm TC dish in 10ml media.
Incubate O/N.
DAY 2
Feed 1x in am
DAY 3
Wash surface of dish with PBS.
Add 1 ml trypsin/ plate.
Incubate 5 min.
Tap plate to detach cells.
Add 10 ml media to plate, transfer all to 50 ml conical
Pellet cells 3 min., 1000rpm
For each embryo, resuspend in 0.5 ml warm media + 0.5 ml warm 2x Freezing Media
Aliquot 1 ml per cryovial
Place cryovials in styrofoam box at -80°C O/N
DAY 4
Transfer cells into liquid nitrogen.
2x Freezing Media
60% DMEM
20% FBS
20% DMSO