- Assessing the Wnt-reactivity of cytonemes of mouse embryonic stem cells using a bioengineering approach.
Assessing the Wnt-reactivity of cytonemes of mouse embryonic stem cells using a bioengineering approach.
STAR protocols (2021-09-28)
Sergi Junyent, Joshua Reeves, Shukry J Habib
PMID34568840
ABSTRACT
These protocols investigate the interaction of cytonemes with localized Wnt. Cell-niche signaling between naive or primed mouse embryonic stem cells (ESCs) and either Wnt-secreting trophoblast stem cells (TSCs) or Wnt signals tethered to microbeads can be scrutinized in vitro. This approach analyzes cytoneme reactivity during Wnt-interaction initiation, Ca2+ transients at Wnt-contacting cytonemes, and subsequent pairing between ESCs and Wnt-sources. This pairing interaction is crucial to synthetic embryogenesis; hence this protocol is effective for in vitro studies of developmental biology. For complete details on the use and execution of this protocol, please refer to Junyent et al. (2020, 2021a, 2021b).
MATERIALS
Product Number
Brand
Product Description
Sigma-Aldrich
Penicillin-Streptomycin, Solution Stabilized, with 5,000 units penicillin and 5mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, With MgCl2 and CaCl2, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
L-Glutamine solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
EDAC, Hydrochloride, EDAC HCl is a water-soluble derivative of carbodiimide useful for conjugating haptens to proteins and polypeptides. Used to modify NMDA receptors and as a condensing agent in peptide synthesis.