EZViable™ Calcein AM Cell Viability Assay Kit (Fluorometric)

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	supplemented with 15% fetal bovine serum (FBS) (Biochrom AG), 1 ng/ml basic fibroblast growth factor (Roche Applied Sciences), 100 µg/ml heparin (Biochrom AG), 5 µg/ml gentamycin and 4 µg/ml puromycin [81] (medium I) at 37°C with a humidified atmosphere of 5% CO2, for 2 days.

Protocol tips
supplemented with 15% fetal bovine serum (FBS) (Biochrom AG), 1 ng/ml basic fibroblast growth factor (Roche Applied Sciences), 100 µg/ml heparin (Biochrom AG), 5 µg/ml gentamycin and 4 µg/ml puromycin [81] (medium I) at 37°C with a humidified atmosphere of 5% CO2, for 2 days.

Publication protocol

Primary cultures of rat BMEC were prepared from 2-week-old Wistar rats. Briefly, meninges were removed from forebrains and gray matter was minced into small pieces in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (DMEM/F12, Biochrom AG, Germany), and digested in DMEM/F12 containing 1 mg/ml collagenase CLS2 (Worthington, USA) for 1.5 h at 37°C. Cell pellet was separated by centrifugation in 20% bovine serum albumin-DMEM/F12 (1000×g, 20 min). Microvessels were further digested with 1 mg/ml collagenase-dispase (Roche Applied Sciences, Switzerland) in DMEM/F12 for 1 h at 37°C. Microvessel endothelial cell clusters were separated on a 33% continuous Percoll (Pharmacia, Sweden) gradient, collected and washed in DMEM/F12 before plating (1.5×105 cells/cm2) onto collagen type IV and fibronectin coated cell culture inserts or multiwell plates. Cultures were maintained in DMEM/F12 supplemented with 15% fetal bovine serum (FBS) (Biochrom AG), 1 ng/ml basic fibroblast growth factor (Roche Applied Sciences), 100 µg/ml heparin (Biochrom AG), 5 µg/ml gentamycin and 4 µg/ml puromycin [81] (medium I) at 37°C with a humidified atmosphere of 5% CO2, for 2 days. On day 3, cells received new medium, which contained all components of medium I except puromycin (medium II).

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