Organotypic Culture Medium
MATERIALS
Reagents
Ascorbic acid (176.12 g/mol; Merck Life Science A92902)
DMEM/F12 (Thermo Fisher 21331020)
FCS (Euroclone ECS5000L)
Glucose (Thermo Fisher A2494001)
Insulin (Merck Life Science L9278)
Milli-Q H2O (sterile)
Pen–strep (Euroclone ECB3001D)
Equipment
Aluminum foil
Filter (0.22 µm) and syringe
METHOD
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1. Prepare a stock solution of 1% ascorbic acid in sterile Milli-Q H2O.
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2. Pass the solution through a 0.22 µm filter to ensure sterility.
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3. Wrap the solution in aluminum foil, and store it for up to 1 mo at 4°C.
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4. Add the following to DMEM/F12 medium under sterile conditions as follows:
| Reagent | Quantity |
|---|---|
| FCS | 10% |
| Milli-Q H2O (sterile) | 10% |
| Insulin | 0.033% |
| Ascorbic acid | 511 µm |
| Pen–strep | 1% |
| Glucose | 0.085% |
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Every time you perform the procedure for organotypic slice production, prepare fresh medium. For periodical changes of medium in the following days, the medium can be prepared and stored for up to 1 wk at 4°C.
