Artificial Cerebrospinal Fluid (ACSF) (1×)

Reagent	Amount to add	Final concentration
Sodium l-lactate	0.448 g	4 mm
Sodium pyruvate	0.220 g	2 mm
Sodium l-ascorbate	0.079 g	0.4 mm
Glucose	4.5 g	25 mm
Add a small amount of H₂O to a 1-L volumetric flask. Add the above reagents and swirl to dissolve. Add 100 mL of Artificial Cerebrospinal Fluid (ACSF) (10×). Fill the flask to ∼950 mL with H₂O. Add 3.95 mL of Calcium Chloride Stock Solution for a final concentration of 1.5 mm CaCl₂. Top off the flask with H₂O to 1 L, and bubble the solution with carbogen for 10 to 20 min (which should resolve any cloudiness from the addition of calcium). Transfer the solution to a 1-L bottle and cap. Store 1× ACSF at 4°C between experiments, and use for up to 1 or 2 d. Do not use the solution if it takes on a pinkish or yellowish color. (1× ACSF can be prepared with different calcium concentrations by changing the added amount of calcium chloride stock solution, and compensating by changing the MgCl₂ so that the total divalent concentration is 2.5 mm. We find it difficult to raise the calcium concentration above 3 mm.)