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Refer to the product datasheet for complete product details.
Briefly, Complete Endothelial Cell Growth Media without VEGF is prepared using the following procedure:
- Add Endothelial Growth Media Supplement without VEGF to Endothelial Cell Growth Base Media
- Store completed media at 2 - 8 °C
Reagents provided with the Endothelial Cell Growth Media (Catalog # CCM029):
- Endothelial Cell Growth Base Media Human
- Endothelial Cell Growth Supplement without VEGF (50X)
Note: The components of this list require different storage/shipping temperatures and will arrive in separate packaging.
Reagents
- HUVECs
- Penicillin-Streptomycin (100X), optional
- TrypLE™ Express
- Phosphate-Buffered Saline (PBS) (Tocris; Catalog # 3156)
Materials
- 75 cm2 tissue culture (T75) flasks
- 15 mL centrifuge tubes
- Serological pipettes
- Pipette and pipette tips
Equipment
- 37°C and 5% CO2 humidified incubator
- Centrifuge (low speed clinical or equivalent)
- Hemocytometer
- Inverted microscope
- Water bath
Reagent Preparation
Endothelial Cell Growth Supplement without VEGF - Thaw the Endothelial Cell Growth Supplement without VEGF (50X) at 2-8 °C or room temperature.
Complete Endothelial Cell Growth Media without VEGF - Add 5 mL of 50X Endothelial Cell Growth Supplement without VEGF to 250 mL of Endothelial Cell Growth Base Media. Add Penicillin-Streptomycin at a 1:100 dilution. Store under sterile conditions at 2-8 °C for up to 2 weeks.
Note: If Penicillin-Streptomycin is not needed for the experiment, it can be omitted.
Culturing of HUVECs
Warm Complete Endothelial Cell Growth Media without VEGF to 37 °C.
Determine the size and number of flasks needed for plating.
Warm the frozen vial of HUVECs until just thawed.
Transfer cells immediately and gently into a 15 mL centrifuge tube > 5 mL of prewarmed Complete Endothelial Cell Growth Media without VEGF.
Centrifuge at 200 x g for 5 minutes.
Resuspend the pellet in prewarmed Complete Endothelial Cell Growth Media without VEGF.
Transfer HUVEC suspension into a T75 flask(s).
Change media the day following thaw.
Monitor cells daily and change media every other day.
Passage the cells at 80-90% confluency.
Subculturing of HUVECs
Warm Complete Endothelial Cell Growth Media without VEGF to 37 °C.
Remove and discard the media from the flasks.
Rinse cells once with sterile 1X PBS.
Digest cells with TrypLE Express for 1-2 minutes at 37 °C.
Tap the side of the flask to aid the detachment of the cells.
Transfer the cell suspension to a 15 mL centrifuge tube containing 8 mL of prewarmed Complete Endothelial Cell Growth without VEGF.
Centrifuge at 200 x g for 5 minutes.
Plate cells (approximately 6.7 x 103 cells/cm2).
Change media the day following thaw.
Monitor cells daily and change media every other day.
Passage the cells at 80-90% confluency.
Endothelial Cell Growth Media without VEGF Summary
Media for the low-serum, VEGF-free expansion of primary endothelial cells.
Key Benefits
- Unique and optimized formulation that promotes endothelial cell growth.
- Free of Vascular Endothelial Growth Factor (VEGF)
- Includes a single supplement featuring key growth factors and small molecules
- 250 mL bottle results in less wasted media
Why Use VEGF-free, low-serum Media to Grow Endothelial Cells?
Primary endothelial cells are commonly used for the study of developmental and disease mechanisms involving the blood vessels, such as angiogenesis. Endothelial cells are phenotypically characterized as CD31+, KDR+, and vWF+ cells.
Inclusion of Vascular Endothelial Growth Factor (VEGF) in endothelial cell media promotes cell proliferation. Under certain experimental conditions, a VEGF-free endothelial media may be beneficial. R&D Systems® Endothelial Cell Growth Media without VEGF is formulated to support the expansion of human primary endothelial cells under low-serum, VEGF-free conditions.
This media has been tested for its ability to support CD31+, KDR+, and vWF+ human umbilical vein endothelial cells growth in vitro.
This kit contains the following reagents to maintain and expand human and mouse endothelial cells in culture:
Containing optimized concentrations of:
- Endothelial Cell Growth Base Media (250 mL)
- Endothelial Cell Growth Supplement without VEGF (50X)
- Human FGF basic
- Human LR3 IGF-1
- Human EGF
- Heparin
- Hydrocortisone
- L-Ascorbic Acid
- Fetal Bovine Serum
Note: The components for this kit require different storage/shipping temperatures and will arrive in separate packaging.
Stability and Storage
Store in the dark at < -20 °C in a manual defrost freezer. Do not use past the expiration date.
Precaution
When handling biohazardous materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.
Limitations
- FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
- The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
- This reagent should not be used beyond the expiration date indicated on the label.
- Results may vary due to variations among cells derived from different donors.