Endothelial Cell Growth Kit-BBE (ATCC^® PCS-100-040^™)

The Endothelial Cell Growth Kit-BBE contains components that when added to Vascular Cell Basal Medium (ATCC PCS-100-030) create a complete cell culture environment for endothelial cells isolated from large vessels. The final concentration for each component in complete endothelial growth medium is as follows:

• Bovine brain extract (BBE): 0.2%
• rh EGF: 5 ng/mL
• L-glutamine: 10 mM
• Heparin sulfate: 0.75 Units/mL
• Hydrocortisone: 1 µg/mL
• Ascorbic acid: 50 µg/mL
• Fetal bovine serum: 2%

1. Obtain one growth kit from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is necessary to warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates, prior to adding to the basal medium.
3. Obtain one bottle of Vascular Cell Basal Medium (475 mL) from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique, and working in a laminar flow hood or biosafety cabinet transfer the volume of each growth kit component, as indicated in Table 1 or 2, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. If using the Endothelial Cell Growth Kit-BBE (ATCC® PCS-100-040), add the indicated volume for each component: 

Component	Volume	Final Concentration
Bovine Brain Extract (BBE)	1.0 mL	0.2%
rh EGF	0.5 mL	5 ng/mL
L-glutamine	25.0 mL	10 mM
Heparin sulfate	0.5 mL	0.75 Units/mL
Hydrocortisone hemisuccinate	0.5 mL	1 µg/mL
Fetal Bovine Serum	10.0 mL	2%
Ascorbic acid	0.5 mL	50 µg/mL

 

Table 2. If using the Endothelial Cell Growth Kit-VEGF (ATCC® PCS-100-041), add the indicated volume for each component:

Component	Volume	Final Concentration
rh VEGF	0.5 mL	5 ng/mL
rh EGF	0.5 mL	5 ng/mL
rh FGF basic	0.5 mL	5 ng/mL
rh IGF-1	0.5 mL	15 ng/mL
L-glutamine	25.0 mL	10 mM
Heparin sulfate	0.5 mL	0.75 Units/mL
Hydrocortisone hemisuccinate	0.5 mL	1 µg/mL
Fetal Bovine Serum	10.0 mL	2%
Ascorbic acid	0.5 mL	50 µg/mL

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of each optional component to be added to the complete growth media is summarized in Table 3.




Table 3. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional)

Component	Volume	Final Concentration
Gentamicin-Amphotericin B Solution	0.5 mL	Gentamicin: 10 µg/mL Amphotericin B: 0.25 µg/mL
Penicillin-Streptomycin-Amphotericin B Solution	0.5 mL	Penicillin: 10 Units/mL Streptomycin: 10 µg/mL Amphotericin B: 25 ng/mL
Phenol Red	0.5 mL	33 µM

 



6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete media is stable for 30 days.