Bronchial Epithelial Cell Growth Kit

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 to the basal medium. 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 Airway Epithelial Cell Basal Medium (485 mL) from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the indicated volume of each growth kit component to the bottle of basal medium using a separate sterile pipette for each. 

Table 1. Bronchial Epithelial Cell Growth Kit Components 

Component	Volume	Final Concentration
HLL Supplement	1.25 mL	HSA 500 µg/mL Linoleic Acid 0.6 µM Lecithin 0.6 µg/mL
L-Glutamine	15 mL	6 mM
Extract P	2.0 mL	0.4%
Airway Epithelial Cell Supplement	5.0 mL	Epinephrine 1.0 µM Transferrin 5 µg/mL T3 10 nM Hydrocortisone 5 µg/mL rh EGF 5 ng/mL rh Insulin .1 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 2.

Table 2. 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.

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