These authors contributed equally to this article.
Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood–brain barrier-specific properties
Article first published online: 21 FEB 2005
Journal of Neurochemistry
Volume 93, Issue 2, pages 279–289, April 2005
How to Cite
Perrière, N., Demeuse, PH., Garcia, E., Regina, A., Debray, M., Andreux, J.-P., Couvreur, P., Scherrmann, J.-M., Temsamani, J., Couraud, P.-O., Deli, M. A. and Roux, F. (2005), Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood–brain barrier-specific properties. Journal of Neurochemistry, 93: 279–289. doi: 10.1111/j.1471-4159.2004.03020.x
- Issue published online: 2 MAR 2005
- Article first published online: 21 FEB 2005
- Received May 26, 2004; revised manuscript received November 24, 2004; accepted November 26, 2004.
- blood–brain barrier;
- in vitro model;
- rat brain microvessel endothelium
One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood–brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 µg/mL puromycin for the first 2 days of culture or 3 µg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 × 10−6 cm/s) and a high electrical resistance (up to 500 Ω × cm2). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood–brain barrier for cellular and molecular biology studies as well as pharmacological investigations.