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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
RÉSUMÉ

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).

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Sérum de veau fœtal, non-USA origin, sterile-filtered, suitable for cell culture
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Diméthylsulfoxyde, ReagentPlus®, ≥99.5%
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Bleu de trypan solution, 0.4%, liquid, sterile-filtered, suitable for cell culture
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L-glutamine solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
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Solution saline dans un tampon phosphate de Dulbecco, With MgCl2 and CaCl2, liquid, sterile-filtered, suitable for cell culture
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N-Hydroxysuccinimide, 98%
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MES, low moisture content, ≥99% (titration)
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Pénicilline-streptomycine, Solution Stabilized, with 5,000 units penicillin and 5mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
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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.
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CNQX, ≥98% (HPLC), solid