STOCK PREPARATIONS:

FBS, chelex-treated
FBS is chelex-treated in batch with Chelex-100 resin (BioRad, cat # 1422832) to remove free Ca⁺⁺. Use 100 g chelex resin per 500 ml FBS. Swell 100 g chelex resin in 400-500 ml distilled water, then titrate to pH 7.4 with HCl while stirring (pH will take a while to stabilize during titration). Filter through Whatman #1 paper. Scrape resin slurry into 500 ml FBS and stir at room temp for 3 hr or at 4℃ overnight. Filter the chelated FBS through Whatman #1 paper and discard the resin slurry. Filter the chelated FBS through a 0.2μm bottle filter to sterilize it. Aliquot sterile, chelated FBS at 20 ml/tube and store at 20℃.
Add 20 ml chelated FBS per 500 ml bottle of EMEM (final conc’n = 4%).

CaCl₂
Prepare a stock of 0.1 M CaCl₂ in sterile water. Filter sterilize with 0.2μm filter.
Add 250μl of 0.1 M CaCl₂ per 500 ml bottle of EMEM (final conc’n = 0.05mM).

Hydrocortisone
Sigma, cat # H0888 Prepare a concentrated stock of 5 mg/ml by dissolving 50 mg hydrocortisone in 10 ml 95% EtOH. Store the concentrated stock at 20℃.
Prepare a 0.2 mg/ml stock by adding 0.4 ml of concentrated stock to 9.6 ml of sterile, serum-free EMEM. Aliquot at 1.0 ml/tube; store at 20℃.
Add 1.0 ml of 0.2 mg/ml hydrocortisone per 500 ml bottle of EMEM (final conc’n = 0.4μg/ml).

Insulin
Bovine Insulin; Sigma, cat # I6634 Prepare a 5 mg/ml stock: Dissolve 100 mg insulin in 20 ml of 0.005 N HCl (prepared by adding 10μl concentrated HCl to 20 ml water).
Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20℃.
Add 0.5 ml of 5 mg/ml insulin per 500 ml bottle of EMEM (final conc’n = 5μg/ml).

Epidermal Growth Factor (EGF)
Recombinant human EGF; GibcoBRL, cat # 13247-051
Prepare a 10μg/ml stock: Dissolve 100μg EGF in 1 ml 0.1% BSA (10 mg ultrapure BSA in 10 ml water). Bring volume to 10 ml with 0.1% BSA. Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20℃.
Add 0.5 ml of 10μg/ml EGF per 500 ml bottle of EMEM (final conc’n = 10 ng/ml).

Cholera Toxin
ICN, cat # 190329 Prepare a concentrated stock of 10^-5M by dissolving 1 mg (1 vial) cholera toxin in 1.18 ml distilled water. Store the stock at 4℃ (do not freeze the concentrated stock).
Prepare a 10^-7M stock by adding 0.1 ml of the concentrated stock to 10 ml EMEM + 4% FBS. Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20℃.
Add 0.5 ml of 10^-7M cholera toxin per 500 ml bottle of EMEM (final conc’n = 10-10 M).

T3 (3,3’,5-triiodo-L-thyronine)
Sigma, cat # T6397
Prepare a concentrated stock of 2x10^-4M by dissolving 13.6 mg T3 in 15 ml 0.02N NaOH, then bringing the volume to 100 ml with PBS. Distribute in 10 ml aliquots and store at 20℃.
Prepare a 2x10^-6M stock by adding 0.1 ml of the concentrated stock to 10 ml PBS. Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20℃.
Add 0.5 ml of 2x10^-6M T3 per 500 ml bottle of EMEM (final conc'n = 2x10^-9 M).

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