12. Add 15ml RPMI 1640 medium
13. Shake the tube, beads will settle
14. Immediately transfer supernatant to new 50ml tube
15. Repeat twice with 15ml RPMI (until 50ml tube full)
16. Spin at 900rpm (tabletop centrifuge) for 5min
17. Resuspend pellet in culture medium and plate on coated dish(es)
18. Human brain microvessels are plated on rat tail collagen and fibronectin coated dishes or glass coverslips.
19. Culture in RPMI 1640 with 10% fetal bovine serum etc (supplementary info)
20. Cultures are incubated at 37oC in a humid atmosphere of 5% CO2 and characterized as described previously (Stins, Gilles, and Kim 1997).
21. The endothelial cultures are more than 95% pure.
22. FACS sorting of pediatric HBMEC was done as previously described (Stins, Gilles, and Kim 1997), yielding more than 99% pure endothelial cell populations.
References
Stins MF, Gilles F, Kim KS,
(1997) Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol. 76, 81-90
Monique
F. Stins, Julie, L Badger, Kwang Sik Kim. Bacterial invasion and transcytosis in transfected human brain endothelial cells. Microbial
Pathogenesis. 30:19-28, 2001.
Stins, MF; Shen, Y; Huang, SH; Gilles, F; Kalra, VK;
Gp120 activates children's brain endothelial
cells via CD4
JOURNAL OF NEUROVIROLOGY, 7(2), 2001, pp. 125 - 134
Supplementary Information (click and see it in next page)
This
Protocol can also be used for Mouse Brain Microvessel Endothelial Cells (MBMECs) isolation with minimum modification.
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