Catalog Number M1011Product Name Mouse brain microvascular endothelial cellsStorage 37°C C0₂ incubator or Liquid NitrogenProduct Format Proliferating cultureCells Number >90% confluent in T25 flask*Caution: Although primary cells are tested pathogen-free, investigators should handle these cells with caution and treat all animal cells aspotential pathogens, since no test procedure can completely guarantee the absence of infectious agents. Proper precautions must be taken toavoid exposure. Always wear proper protective equipment (Gloves, safety glasses, etc.) when handling these materials. We recommendfollowing the universal procedures for handling products of animal origin as the minimum precaution against contamination.GENERAL INFORMATIONMouse brain microvascular endothelial cells were isolated from E18 embryonic brain cortexmicrovessels of Balb/C mouse and transformed by transfected with SV40 middle T antigen. The cells areshipped in proliferating culture or frozen vial with a confluence of > 90% (the cells are provided atpassage 4-6). Cells are tested negative for common experimental animal pathogens, and mycoplasma invitro. ENDO-Growth medium (EGM-2102) containing 5% serum and growth supplement isrecommended for the expansion of these can be propagated to passage 6 and beyond without losingtheir morphologic and phenotypic characteristics. When you receive the cells, leave the flask in 37°C C02incubator for 1 hour first and then replace the transport medium with fresh ENDO-Growth medium(EGM-2102). Let the cells grow for 24 hours before subculture.CELL CHARACTERIZATIONPECAM1 >95% positive by immunofluorescenceVE-Cadherin >95% positive by immunofluorescenceMouse brain microvascular endothelial cells Negative for mycoplasmaPRODUCT USE AND SHIPPING STATUSProduct Use Mouse brain microvascular endothelial cells arefor research onlyShipping Status Proliferating culture inT25 flask
T25 flask*Coating T25 flasks. Add 2 ml AlphaBioCoat (AC001) into 3- T25 flasks and ensure entire interiorsurface is coated with the solution. After 30 minutes, dispose of AlphaBioCoat (AC001) byaspiration. Gently rinse and aspirate the flask with Phosphate Buffer Solution (1XPBS-001). Theflask is now ready for use (no need for overnight incubation when coated with AC001). Addfresh media to flask, if color changes from pink to yellow, discard the media, and add freshmedia to each flask.1. Inspect to make sure Flask is at 90% confluence, if not remove transport media, and add 5ml offresh media to the flask. Place flask in 37°C incubator until cells are at 90% confluence. Changemedia every 2 days.2. If flask is at 90% confluence, aspirate transport media from flask.3. Rinse T25 flask containing cells with 5 ml 1XPBS (1XPBS-001).4. Gently aspirate out the PBS after rinsing, and discard.5. Add 2ml of RT trypsin/ EDTA to T25 flask containing cells (ensure entire interior surface iscovered.6. Place T25 flask containing cells into 37°C incubator for 1 or 2 minutes (cells will normally comeoff of the surface within 1 or 2 minutes).7. Suspend the cells with 15ml of ENDO-Growth medium (EGM-2102) and transfer equally into 3pre-coated T25 flasks (the cells are now at a subculture ratio of 1:3.8. There is no need to spin cells during subculture.9. Proliferating cell culture: ENDO-Growth medium (EGM-2102) should be changed every 2 days.The cells normally become confluent within 7 days (when split at a 1:3 ratio)10. Use ENDO- Basal media (EBM-002) containing 0.5% FBS to induce quiescent cells (after 18-24hours).