Endothelial Cell Growth Medium

Reagent	Volume	Final concentration
Neurobasal medium (Gibco 21103-049)	18.9 mL	95%
SATO Supplement, NB-based (100×)	200 µL	1×
T3 stock (4 µg/mL)	200 µL	40 ng/mL
l-Glutamine (200 mm; Gibco 25030-081)	200 µL	2 mm
Sodium pyruvate (100 mm; Gibco 11360-070)	200 µL	1 mm
Penicillin/streptomycin (Gibco 15140-122)	200 µL	100 U/mL (penicillin) 100 µg/mL (streptomycin)
Insulin (0.5 mg/mL)	200 µL	5 µg/mL
NAC stock (5 mg/mL)	20 µL	5 µg/mL
Puromycin (10 mg/mL; Sigma P9620)^a	2 µL	1 µg/mL
Forskolin stock (4.2 mg/mL)^a	20 µL	4.2 µg/mL
FCS (100%; heat-inactivated for 30 min at 55°C; Gibco 16000-036)^a	100 µL	0.5%
Forskolin stock (4.2 mg/mL)^b	20 µL	4.2 µg/mL
bFGF (50 µg/mL)^b	20 µL	50 ng/mL
FCS (100%; heat-inactivated for 30 min at 55°C; Gibco 16000-036)^b	100 µL	0.5%

Filter the medium through a prerinsed 0.22-µm filter into a conical tube. Store at 4°C.
^aIf the endothelial cells come from rodents older than P20, include these components in the medium on Days 1, 2, and 3 of culture to kill other types of cells.
^bIf the endothelial cells come from rodents older than P20, include these components in the medium beginning on Day 4 of culture to promote brain endothelial cell proliferation. If the cells come from rodents P20 and younger, include these components in the medium from Day 1 onward.