Endothelial Cell Growth Media without VEGF
R&D Systems, Inc. a Bio-Techne Brand
Key Product Details
Assay Procedure
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.

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