Technical Reference #1889

1889.

Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis William P. Daley; Kathryn M. Gulfo; Sharon J. Sequeira; Melinda Larsen, State University of New York - University at Albany, Developmental Biology, 336(1889), (2009)

Keywords:
Submandibular gland; Branching morphogenesis; ROCK; MLC2; Fibronectin; Proliferation; Basement membrane; Actomyosin contractility

Materials & Methods:
Mouse SMGs were dissected from timed-pregnant female mice (strain CD-1 Charles River Laboratories) at either embryonic day 12 (E12 1 bud) or E13 (4–5 buds) with the day of plug discovery designated as E0 following protocols approved by the University at Albany IACUC committee. SMGs were microdissected from mandible slices and cultured as previously described (Rebustini and Hoffman 2009; Sakai and Onodera 2008). Briefly mandible slices were removed from embryos with a sterile scalpel and from these SMGs were microdissected using sterile forceps under a dissecting microscope (SMZ645 Nikon). The SMGs were cultured on 13 mm 0.1 μm Nuclepore Track-Etch membrane filters (Whatman) floating on 200 μL of 1:1 DMEM/Ham's F12 Medium (F12) lacking phenol red (Invitrogen) in glass-bottom 50 mm microwell dishes (MatTek Corporation). The medium was supplemented with 50 μg/mL transferrin 150 μg/mL L-ascorbic acid 100 U/mL penicillin and 100 μg/mL streptomycin to make complete DMEM/F12 medium.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
mouse SMG-derived epithelial cell line