Catalog Number B1021Product Name GFP expressing bovine aortic endothelial cellsStorage 37°C C02 incubatorProduct 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 INFORMATIONGFP expressing bovine aortic endothelial cells were transfected using lentri-virus expressing GFP usingpuromycin. The cells are shipped in proliferating culture or frozen vial with a confluence of > 90% (thecells are provided at passage 2). ENDO-Growth medium (EGM-2102) containing 5% serum and growthsupplement is recommended for the expansion of these can be propagated to passage 6 and beyondwithout losing their morphologic and phenotypic characteristics. When you receive the cells, leave theflask in 37°C C02 incubator for 1 hour first and then replace the transport medium with fresh ENDOGrowth medium (EGM-2102). Let the cells grow for 24 hours before subculture.CELL CHARACTERIZATIONPECAM1 >95% positive by immunofluorescenceVE-Cadherin >95% positive by immunofluorescenceGFP Expressing bovine aortic endothelial cells Negative for mycoplasmaPRODUCT USE AND SHIPPING STATUSProduct Use GFP expressing bovine aortic endothelial cells arefor research onlyShipping Status Proliferating culture in T25 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).