3 Preparation of Media
29
Comments
The original GMA formulation contains penicillin and streptomycin. If
the possibility of contamination by bacteria is high, use these antibiotics; otherwise, they can be omitted from the solution.
Subprotocol 4
Preparation of Media Containing Water-Insoluble Substances
Some cell culture media contain water-insoluble substances, such as lipids. These substances are usually dissolved in some organic solvent, and
are then dispersed in the media as a fine emulsion. In general, organic
solvents are toxic to cells at high concentrations, and, therefore, incorporation of such a solvent in the culture medium should be minimized. As
an example of these type of media, the preparation of the serum-free
medium of Maiorella et aI. (1988) is given below.
Materials
- Chemical balance (capacity 200 g, readability 0.1 mg)
Equipment
- Magnetic stirrer (one with an installed heating system is preferable)
- pH meter (readability O.oI)
- 10,000 MW cut-off PMlO hollow fiber cartridge (Amicon Corp.)
- Cod liver fatty acid methyl ester
Chemicals
- Tween-80
- Cholesterol
- u-Tocopherol acetate
- Ethyl alcohol
- IPL-41 medium (Weiss et aI., 1981; for the composition of IPL-41, see Solutions
Appendix 1).
IPL-41 medium can be prepared in the same manner as Grace's medium except for the inclusion of trace metals. IPL-41 trace metals will
be stored as a 1000-fold concentrated stock solution (4 mg ZnCl z , 2 mg
MnClz·4HzO, 20 mg CuClz·2HzO, 4 mg (NH4)M070Z4·4HzO, 5 mg
CoCl z ·6H z O and 55 mg FeS0 4 ·7H z O per 100 ml distilled water). The
stock solution can be stored in a refrigerator for several months.
- A 10% aqueous solution of Pluronic polyol F-68
- A 10% TC-yeastolate (Difco Laboratories)
Dissolve 100 g TC-yeastolate in 1,000 ml distilled water. Ultrafilter the
solution through a 10,000 MW cut-off PM 10 hollow fiber cartridge.
29
Comments
The original GMA formulation contains penicillin and streptomycin. If
the possibility of contamination by bacteria is high, use these antibiotics; otherwise, they can be omitted from the solution.
Subprotocol 4
Preparation of Media Containing Water-Insoluble Substances
Some cell culture media contain water-insoluble substances, such as lipids. These substances are usually dissolved in some organic solvent, and
are then dispersed in the media as a fine emulsion. In general, organic
solvents are toxic to cells at high concentrations, and, therefore, incorporation of such a solvent in the culture medium should be minimized. As
an example of these type of media, the preparation of the serum-free
medium of Maiorella et aI. (1988) is given below.
Materials
- Chemical balance (capacity 200 g, readability 0.1 mg)
Equipment
- Magnetic stirrer (one with an installed heating system is preferable)
- pH meter (readability O.oI)
- 10,000 MW cut-off PMlO hollow fiber cartridge (Amicon Corp.)
- Cod liver fatty acid methyl ester
Chemicals
- Tween-80
- Cholesterol
- u-Tocopherol acetate
- Ethyl alcohol
- IPL-41 medium (Weiss et aI., 1981; for the composition of IPL-41, see Solutions
Appendix 1).
IPL-41 medium can be prepared in the same manner as Grace's medium except for the inclusion of trace metals. IPL-41 trace metals will
be stored as a 1000-fold concentrated stock solution (4 mg ZnCl z , 2 mg
MnClz·4HzO, 20 mg CuClz·2HzO, 4 mg (NH4)M070Z4·4HzO, 5 mg
CoCl z ·6H z O and 55 mg FeS0 4 ·7H z O per 100 ml distilled water). The
stock solution can be stored in a refrigerator for several months.
- A 10% aqueous solution of Pluronic polyol F-68
- A 10% TC-yeastolate (Difco Laboratories)
Dissolve 100 g TC-yeastolate in 1,000 ml distilled water. Ultrafilter the
solution through a 10,000 MW cut-off PM 10 hollow fiber cartridge.