SATO Supplement, NB-based (100×)

• 1. Prepare the following stock solutions (these should be made fresh; do not reuse).

  • Combine 2.5 mg of progesterone (Sigma-Aldrich P8783) and 100 µL of ethanol to make a progesterone stock solution.

  • Combine 4.0 mg of sodium selenite (Sigma-Aldrich S5261), 10 µL of 1 n NaOH, and 10 mL of Neurobasal (NB, Gibco 21103-049) to make a sodium selenite stock solution.

• 2. Add the following to 80 mL of Neurobasal medium:

  Reagent	Quantity	Final concentration in medium (1×)
  BSA (Sigma-Aldrich A4161)	800 mg	100 µg/mL
  Transferrin (Sigma-Aldrich T1147)	800 mg	100 µg/mL
  Putrescine dihydrochloride (Sigma-Aldrich P5780)	128 mg	16 µg /mL
  Progesterone stock solution	20 µL	60 ng/mL (0.2 µm)
  Sodium selenite stock solution	800 µL	40 ng/mL

• 3. Mix well, and filter-sterilize through a prerinsed 0.22-µm filter. Make 200-µL or 800-µL aliquots, and store at −20°C.

• © 2013 Cold Spring Harbor Laboratory Press