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Catalog Number GF8Product Name GFP Expressing human retinal microvascular endothelialcells (GFP- HRMECs)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 INFORMATIONGFP expressing human retinal microvascular endothelial cells were initiated by elutriation fromdissociated normal human retinal tissue and transfected with GFP- lentiviral particles at passage 1.Puromycin resistant GFP-HNDFs were selected and shipped in proliferating culture or frozen vial with aconfluence of > 90% (cells are providing at passage 4-5). ENDO-Growth medium (EGM-001) containing5% serum and growth supplement is recommended for culture. Cells have an average populationdoubling level >16 when cultured. When you receive the cells, leave the flask in 37°C C02 incubator for 1hour. Then, replace the transport medium with fresh ENDO-Growth medium(EGM-2102). Let the cellsgrow for 24 hours before subculture.CELL CHARACTERIZATIONCytoplasmic VWF/ factor VIII >95% positive by immunofluorescenceCytoplasmic uptake of Di-I-Ac-LDL >95% positive by immunofluorescenceCytoplasmic PECAM1 >95% positive by immunofluorescenceGFP Expressing retinal microvascular endothelialcells are negative forHIV-1, HBV, HCV, and mycoplasmaPRODUCT USE AND SHIPPING STATUSProduct Use GFP Expressing retinal microvascular endothelialcells 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 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-001) 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 cells culture: ENDO-Growth medium (EGM-001) 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).