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Complete Growth Medium
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- Obtain one growth kit from the freezer; make sure that the caps of all containers are tight.
- 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.
- Obtain one bottle of Fibroblast Basal Medium (480 mL) from cold storage.
- Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
- 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 Fibroblast Growth Kit–Serum-Free (ATCC® PCS-201-040), add the indicated volume for each component in the order shown.
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Component
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Volume
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Final Concentration
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L-glutamine
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18.75 mL
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7.5 mM
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Hydrocortisone Hemisuccinate
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0.5 mL
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1 μg/mL
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HLL Supplement
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1.25 mL
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HSA 500 μg/mL
Linoleic Acid 0.6 μM
Lecithin 0.6 μg/mL
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rh FGF b
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0.5 mL
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5 ng/mL
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rh EGF / TGF b-1 Supplement
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0.5 mL
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5 ng/mL
30 pg/mL
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rh Insulin
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0.5 mL
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5 μg/mL
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Ascorbic acid
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0.5 mL
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50 μg/mL
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Table 2. If using the Fibroblast Growth Kit–Low Serum (ATCC® PCS-201-041), add the indicated volume for each of the following components:
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Component
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Volume
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Final Concentration
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rh FGF b
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0.5 mL
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5 ng/mL
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L-glutamine
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18.75 mL
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7.5 mM
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Ascorbic acid
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0.5 mL
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50 μg/mL
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Hydrocortisone Hemisuccinate
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0.5 mL
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1 μg/mL
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rh Insulin
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0.5 mL
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5 μg/mL
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Fetal Bovine Serum
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10.0 mL
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2%
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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)
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Component
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Volume
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Final Concentration
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Gentamicin-Amphotericin B Solution
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0.5 mL
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Gentamicin: 10 µg/mL
Amphotericin B: 0.25 µg/mL
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Penicillin-Streptomycin-Amphotericin B Solution
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0.5 mL
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Penicillin: 10 Units/mL
Streptomycin: 10 µg/mL
Amphotericin B: 25 ng/mL
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Phenol Red
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0.5 mL
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33 µM
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- 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.
- Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.
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