Motor Neuron Growth Medium

• 1. Add Neurobasal Medium (Gibco/Life Technologies 21103) to the top of a sterile filter unit.

• 2. Add the following:

  Reagent	Final concentration
  B-27 Serum-Free Supplement (Gibco/Life Technologies 17504-044)	1×
  SATO supplement (100×)	1×
  Insulin stock (0.5 mg/mL)	5 μg/mL
  Sodium pyruvate (Cambrex 13-115E or Gibco/Life Technologies 11360-070)	1 mm
  l-Glutamine (Mediatech 25-005-CI)	2 mm
  T3 stock (4 μg/mL)	40 ng/mL
  Mouse laminin (Gibco/Life Technologies 23017-015)	1 μg/mL
  Isobutylmethylxanthine (RBI A-007)	2.2 μg/mL
  Forskolin stock (4.2 mg/mL)	417 ng/mL
  NAC stock (5 mg/mL)	5 μg/mL
  Penicillin-streptomycin (Gibco/Life Technologies 15140-122)	100 U/mL (penicillin) 100 μg/mL (streptomycin)

  Recently, some lots of B-27 supplement have been toxic; alternatives include B-27 minus antioxidants or NS21 supplement (R&D Systems AR008).

• 3. Filter-sterilize.

• 4. Add the following:

  Reagent	Final concentration
  BDNF stock (10 μg/mL)	10 ng/mL
  CNTF stock (10 μg/mL)	10 ng/mL
  GDNF stock (10 μg/mL)	10 ng/mL

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