Microvascular Endothelial Cell Growth Kit-BBE

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 Microvascular Endothelial Cell Growth Kit-BBE (ATCC^® PCS-110-040), 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	25.0 mL	5%
Ascorbic acid	0.5 mL	50 µg/mL

 

       Table 2. If using the Microvascular Endothelial Cell Growth Kit-VEGF (ATCC^® PCS-110-041), 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	25.0 mL	5%
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 growth 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.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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