Dissect cranial facial regions, wing buds and hind limb buds
from HH stage 22-24
embryos in sterile Howard's
Do not use stage 25 or
with sterile Ca++,
facial cells and limbs from 5 embryos in 10ml 0.1% trypsin
7072IL), 0.1% collagenase
(Sigma C-6885) in CMFTy.
Incubate at 37oC
with gentle shaking for 1 hour.
Gently pipette up and down
with cut off transfer pipette to disperse cells.
Add 0.5 ml newborn calf serum
(NCS) to stop protease digestion.
Transfer to 10ml tube
Centrifuge at 1500 rpm for 10
Discard supernatant. Gently
flick to resuspend cells.
sterile hood, add 10% NCS i n CMFTy t o 0. 3 - 0.35 ml final
Remove small drops of cells.
Leave remaining cells in hood.
Dilute 10ml cells, 10ml Trypan blue solution with 80ml
Adjust cell concentration to 2 x 10^7 cells/ml with 10% NCS
Transfer 15 micro liter
drops to center of wells on a 24 well plate. Allow to set