MEDIA FOR EMBRYO CULTURE AND MANIPULATION

 

M16 Medium (Protocol obtained from Karen Austen-Reed from SS Tan Laboratory, Anatomy Department)

For oocyte maturation and routine culture of mouse embryos, M16 culture medium is used. This medium is unable to maintain its own pH and must therefore by used in conjuction with an incubator buffered with 5% CO2. The CO2 maintains the required pH level of the medium.

Compound	mM	Mol. Wt.	g/500ml
NaCl	94.66	58.45	2.766
KCl	4.78	74.557	0.178
KH2PO4	1.19	136.091	0.081
MgSO4-7H2O	1.19	246.5	0.146

A OR

 

MgSO4 (anhydrous)			0.072
Na Lactate	23.28	112.1	1.305 (powder form) or 1.69ml (60% syrup
Glucose	5.56	179.86	0.5

Penicillin/Streptomycin - use at 1/100 (Tissue culture Pen/Strep)

Phenol red 0.005

B

NaHCO3 25.0 84.02 1.051

C Na Pyruvate 0.33 110.0 0.018

D CaCl2-2H2O 1.71 147.2 0.126

BSA (Bovine Serum Albumin) 4mg/ml

Method

Weigh out all of stock "A" (except Pen/Strep and Na lactate) into a measuring cylinder and make up to 90ml with "Travenol" water. Add Pen/Strep to cylinder and then Na lactate (Note: the Na lactate is quite viscous - by heating it up to ~37oC prior to use it can be more easily and accurately pipetted). Make up to 100ml with "Travenol" water and pour into 500ml bottle.

Weigh out stock "B" (Phenol red, NaHCO3) components into a measuring cylinder. Make up to 100ml with "Travenol" and pour into 500ml bottle. Weigh stock "C" (Na Pyruvate) into measuring cylinders, make up to 100ml with "Travenol" water and pour into 500ml bottle. Weigh stock "D" (CaCl2-2H2O) into measuring cylinder, make up to 100ml with "Travenol" water and pour into 500ml bottle. Add about 100ml of "Travenol" water to bring the total volume to 500ml. Be sure that the components of each stock have dissolved properly before adding to the 500ml bottle. Also be sure to add stocks in their alphabetical order to avoid precipitation of some ingredients.

Make 50ml aliquots of this solution and freeze them. Use one 50ml aliquot at a time by aliquoting it into tubes of 9 ml each, then refrigerate until use. The osmolarity should be 288-292 mosmol.

Before use, lightly gas with CO2, then add 1 ml of FCS (foetal calf serum) to a 9 ml aliquot of M16. Then sprinkle 36mg of BSA (4mg/ml M16) on top and allow to dissolve - do not shake up or stir. Then Filter-sterilize this mix using a 12ml syringe with an acrodisc.

DMEM WITH HEPES

This medium is used for manipulations that are performed on the mouse embryos whilst out of the incubator. Since there is not 5% CO2 present to help maintain the pH, this medium contains HEPES to keep the pH constant.

Ingredient % of Final Volume

1 x DMEM with 20mM HEPES buffer 80.8%

Penicillin/Streptomycin (5,000 i.u/ml/5,000ug/ml) 1%

L-glutamine (200mM, 100x) 1%

non-essential amino acid (100x) 1%

nucleosides (100x) 1%

b-mercaptoethanol (0.1M) 0.2%

Foetal calf serum 15%

This recipe is made up fresh each week (the same stocks of ingredients may be used). An alternative to DMEM with HEPES is M2, the recipe for which can be found in Bridgette Hogan's Book, "Manipulating the Mouse Embryo".