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Catalog Number RFP2Product Name RFP Expressing Human Brain Microvascular EndothelialCells (RFP-HBMVPCs)Storage 37°C C0₂ incubatorProduct Format Proliferating cultureCells Number >90% confluent in T25 flask*Caution: The handling of human derived products has the potential to be biologically hazardous. All Cell strains tested negative for HIV, HBV,and HCV DNA in diagnostic tests. Proper precautions must be taken to avoid exposure. Always wear proper protective equipment (Gloves,safety glasses, etc.) when handling these materials. We recommend following the universal procedures for handling products of human originas the minimum precaution against contamination.GENERAL INFORMATIONRFP expressing human Brain Microvascular Endothelial Cells were isolated from normal human braintissue and transfected with RFP-Expressing lentiviral particles at passage 1. Puromycin resistant, RFPHBMVPCs is shipped in proliferating culture or frozen vial with a confluence of > 90% (cells are provide@ passage 4-5). Endo-Growth medium (EGM2102) containing 5% serum and growth supplement isrecommended for culture. Cells have an average population doubling level >10 when cultured. Whenyou receive the cells, leave the flask in 37°C C02 incubator for 1 hour. Then, replace the transportmedium with fresh Endo-Growth medium (EGM2102). Let the cells grow for 24 hours beforesubculture.CELL CHARACTERIZATIONCytoplasmic Alpha-Actinin Filaments >80% positive by immunofluorescenceCytoplasmic Desmin Intermediate Filaments >80% positive by immunofluorescenceCytoplasmic Uptake of Di-I-Ac-LDL <2% Positive by immunofluorescenceCytoplasmic VWF/ Factor VIII <2% positive by immunofluorescenceRFP expressing human brain microvascularendothelial cells are negative forHIV-1, HBV, HCV, and mycoplasmaPRODUCT USE AND SHIPPING STATUSProduct Use RFP expressing human brain microvascularEndothelial cells are for research use 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.4. Gently aspirate out the PBS after rinsing, and discard.5. Add 2ml of to T25 flask AlphabioDetach Cell Detachment Solution (ADF001) containing cells(ensure entire interior surface is covered).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 and transfer equally into 3 pre-coated T25flasks (the cells are now at a subculture ratio of 1:3.8. There is no need to spin cells during subculture.9. Proliferating cells culture: Endo-Growth medium (EGM2102) 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 18-24