Dec 19, 2023

Trophoblast Organoid Media (TOM)

This protocol is a draft, published without a DOI.
  • 1Duke University
vfarmer Farmer
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Protocol Citation: vfarmer 2023. Trophoblast Organoid Media (TOM). protocols.io https://dx.doi.org/
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 15, 2023
Last Modified: December 19, 2023
Protocol  Integer ID: 92353
Keywords: flipped to, media
Abstract
Media is also used with flipped TOs. Media can be stored at 4C and used within 1 month.
Recipe
In a 50 mL conical add 40 mL of F12/DMEM. Mix in the following components:
B27 1 mL

stock is 10X, final is 1X

supplier: ThermoFisher (Gibco), cat#: 17504044
N-Acetyl-L-Cystine (NAC) 625 µL

stock is 100 mM in H20, final is 1.25 mM

supplier: Sigma, cat#: A9165

N2 500 µL

stock is 10X, final is 1X

supplier: ThermoFisher (Gibco), cat#: 17502048
L-Glutamine 500 µL

stock is 200 mM, final is 200 mM

supplier: ThermoFisher (Gibco), cat#: A2916801
Nicotinamide (NTM) 500 µL

stock is 1 M in H20, final is 10 mM

supplier: Sigma-Aldrich, cat#: N0636
Primocin 100 µL

stock is 50 mg/ml, final is 100 µg/ml

supplier: Inviogen, cat #: NC9141851
Y-27632 100 µL

stock is 2.5 mM in H20, final is 5 µM
hEGF 25 µL

stock is 100 µg/ml in H20, final is 50 ng/ml

supplier: Peprotech, cat#: AF-100-15
hFGF2 25 µL

stock is 200 µg/ml in 5mM Tris pH 7.6, final is 100 µg/ml

supplier: Peprotech, cat#: 100-18C
hHGF 25 µL

stock is 100 µg/ml in H20, final is 50 ng/ml

supplier: Peprotech, cat#: 100-39
R-spondin1 25 µL

stock is 160 µg/ml in PBS+0.1% BSA, final is 80 µg/ml

supplier: Bio-techne, cat#: 4645-RS
PGE2 25 µL

stock is 5 mM in acetone, final is 2.5 µM

supplier: Sigma, cat#: P0409
A83-01 5 µL

stock is 5 mM in DMSO, final is 500 nM

supplier: Tocris, cat#: 2939
CHIR99021 5 µL

stock is 15 mM in DMSO, final is 1.5 µl

supplier: Tocris, cat#: 4423
FBS 5 mL

supplier: Cytiva, cat# SH30088.03HI