PRIMARY CULTURE AND IDENTIFICATION OF MOUSE BRAIN MICROVASCULAR ENDOTHELIAL CELLS

Brain microvascular endothelial cells are the basic components of blood-brain barrier. Many neurological diseases related to loss blood brain barrier (BBB) function. Isolating and culturing primary BMVEC is an important means study function regulation BBB in vitro.
 To establish a method for isolation culture mouse (BMECs). The 7-10-days mice were sacrificed by neck removal, cranial cavity was opened, aseptically removed cerebral cortex retained. extract segments, underwent three D-Hank solution rinses. It then homogenized, twice digested enzyme, centrifuged using Bovine Serum Albumin (BSA) gradient. For culture, segments injected into gelatin-coated plates DMED complete media. When cell density reaches 90 %, media removed, given two PBS washes. A new medium introduced after adding 1ml trypsin-EDTA digest 2–5 minutes passage.
 Cell rinsed pre-cooled 95 % ethanol added 20 passaged had grown 80–90 their original size. After third wash, 1 ml factor VIII antibody wells, where it left 4 hours at 37 °C. FITC-labeled rabbit anti-mouse volume mL added. Under confocal laser microscope, examined taken pictures of. outcome demonstrates positive expression marker associated antigen.
 create suspension, monolayer-growing chosen. 100 L suspension used inoculate each well 96-well plates, grown. On days 1, 2, 3, 4, 5, 6, 7, 180 μL DMEM MTT simultaneously 4-hour culture. dimethyl sulfoxide (DMSO), well's absorbance value (A value) measured 492 nm. that growth peak attained between 6 8 days.
 This can successfully isolate BMECs, which lay foundation BMEC vitro.