Timed pregnant mice were sacrificed on gestational day E8.5, and
individual embryos were staged according to somite number (ss). Experiments were
performed using embryos between 4–12ss. For E8.5 foregut cultures, the
neural headfolds and forebrain were usually removed, as well as tissue caudal to
the anterior intestinal portal (AIP). For foregut cultures in which liver and
pancreas formation were studied, embryos were cut 4 somites caudal to the AIP to
ensure that dorsal pancreas progenitors were captured within the region being
cultured (Angelo et al., 2012 (link)). Regions
containing the foregut were dissected in Hank's balanced salt solution
(HBSS), explanted onto 8 µm pore Whatman Nucleopore Track-Etch Membranes
(Millipore) or Transwell Collagen Filters (Costar) and cultured for 2–5
days in DMEM (Gibco) +20% fetal bovine serum (FBS; Sigma). In some
experiments 20% FBS was replaced with 20% KnockOut Serum
Replacement (KOSR; Gibco). The VEGF receptor inhibitors Ki8751 and SU5416 (both
from Calbiochem) were used at 10 µM. These compounds have been shown to
effectively inhibit VEGF signaling at this concentration (Fong et al., 1999 (link);Havrilak
and Shannon, 2015b ; Kubo et al.,
2005 ). In all experiments DMSO served as a vehicle control.
individual embryos were staged according to somite number (ss). Experiments were
performed using embryos between 4–12ss. For E8.5 foregut cultures, the
neural headfolds and forebrain were usually removed, as well as tissue caudal to
the anterior intestinal portal (AIP). For foregut cultures in which liver and
pancreas formation were studied, embryos were cut 4 somites caudal to the AIP to
ensure that dorsal pancreas progenitors were captured within the region being
cultured (Angelo et al., 2012 (link)). Regions
containing the foregut were dissected in Hank's balanced salt solution
(HBSS), explanted onto 8 µm pore Whatman Nucleopore Track-Etch Membranes
(Millipore) or Transwell Collagen Filters (Costar) and cultured for 2–5
days in DMEM (Gibco) +20% fetal bovine serum (FBS; Sigma). In some
experiments 20% FBS was replaced with 20% KnockOut Serum
Replacement (KOSR; Gibco). The VEGF receptor inhibitors Ki8751 and SU5416 (both
from Calbiochem) were used at 10 µM. These compounds have been shown to
effectively inhibit VEGF signaling at this concentration (Fong et al., 1999 (link);
and Shannon, 2015b
2005