Modified Barth’s medium: 1L recipe
5.14g NaCl
75mg KCl
0.2g NaHCO3
0.2g MgSO4•7H2O
60mg CaCl2•2H2O
80mg Ca(NO3)2•4H2O
2.38g HEPES, free-acid
Adjust the pH to 7.4 with NaOH. Autoclave.
CMF-MBS
Buffer
This is Ca2+/Mg2+-free version of MBS-H. 500ml recipe :
2.57g NaCl
37.5mg KCl
0.1g NaHCO3
1.19g HEPES, free-acid
Adjust the pH to 7.4 with NaOH. Autoclave.
1% agarose/1x Barth’s plates
as many as the number of samples
1% agarose/CMF-MBS plates
as many as the number of samples
E-Cadherin/Fc chimera
Reagent
Sigma
E-2278
Dissolve 0.1mg of solid E-cadherin in 100ul of 1x MBS-H containing 40% glycerol to make 100x master stock (1.0mg/ml). Store it in –20C. Make 1/100 dilution with 1x MBS-H to make 10ug/ml working solution just before you use. You need 200ul per samples of this working solution.
Fibronectin, 0.1% solution
Reagent
Sigma
F-1141
This pre-made solution can be used as 50x stock. Make 1/50 dilution with 1x MBS-H make 20ug/ml working solution just before you use. You need 200ul per samples of this working solution.
I do NOT recommend using solid Fibronectin, because it is difficult to dissolve at 0.1% concentration to make the stock solution. Although it is more expensive, this pre-made 0.1% solution is better to obtain consistent results.
Ogata, S., Cho, K. W. Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay. J. Vis. Exp. (3), e187, doi:10.3791/187 (2007).