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Recipes (old):

1. Nutrient Mixture F12; 5% FCS Medium (FOR 1 LITER)

          Materials:

• Nutrient Mixture F12 COONS Modification, powder
• Sigma Catalog Number F6636 10 x 1L = $26.80 (This is the only commercially available medium tested so far that provides growth conditions for WIF-B cells similar to that of the original special formulation by Gibco). 
• Distilled Deionized Water, DDH₂O, > 10 megohm-cm
• Sodium Bicarbonate, NaHCO3, M.W. 84.01, Baker #3605-05
• CO₂ Gas
• 100x SPF Antibiotic Stock (see protocol recipe IV)
• 1000x HAT Stock (see protocol recipe II)
• Fetal Bovine Serum (FBS)
• Sterile 0.22 m m Filter set

          Protocol for 1 Liter:

1. Add 1 1L package of Nutrient Mixture F12 Medium powder to 900 ml DDH2O.
2. Add 2.7 g of Sodium Bicarbonate
3. Add 10 ml 100x SPF Stock.
4. Add 1 ml 1000x HAT Stock.
5. Bring volume up to 1 L with DDH2O, and mix until dissolved.
6. CO2 gas to medium to attain the pH of a 6.8 (after sterile filtration pH should be ~7.0, the color should be a salmon-pink {medium can be pH’ed with HCl also}).
7. Add 5% FBS to medium.
8. Sterile filter (0.22 m m) the medium into the appropriate labeled (sterile) bottles, and then incubate sample at 37° C overnight to check for sterility. Store at 4° C to 8° C.

          Alternate Protocol for 1 Liter:

1. Add 1 1L package of Nutrient Mixture F12 Medium powder to 900 ml DDH₂O.
2. Add 2.7 g of Sodium Bicarbonate.
3. Add 5 ml reconstituted Penicillin G-Streptomycin (sigma cat. No. P-3539 or P-0781)
4. Add 90 mg Penicillin G (Sigma cat. No. P-3032)
5. Add 2 ml Amphotericin B (Sigma cat. No. A-2942)
6. Add 1 ml 1000x HAT Stock
7. Bring volume up to 1 L with DDH₂O, and mix until dissolved.
8. CO₂ gas to medium to attain the pH of a 6.8 (after sterile filtration pH should be ~7.0, the color should be a salmon-pink {medium can be pH’ed with HCl also}).
9. Add 5% FBS to medium.
10. Sterile filter (0.22 m m) the medium into the appropriate labeled (sterile) bottles, and then incubate sample at 37° C overnight to check for sterility. Store at 4° C to 8° C.

2. 100x SPF Stock

          Materials:

• "Penicillin G, 10,000 Unit/ml – Streptomycin, 10 mg/ml" 20 ml vial, Sigma Cat. #P-3539 or P-0781 can substitute.
• Penicillin G, 1,670 Units/mg, Sigma Cat. #P-3032
• 500x Amphotericin B (Fungizone) Stock (Sigma Cat. No. A-2942)

          Protocol:

1. Add 8 ml of 500x Amphotericin B (Fungizone) stock and 12 ml of DDH₂O to the 20 ml vial of "Penicillin G, 10,000 Unit/ml – Streptomycin, 10 mg/ml" (P-3539).
2. Add 360 mg of Penicillin G, 1,670 Units/mg, to the 20 ml dilution.
3. Bring the volume up to 40 ml with DDH2O and sterile filter (0.22 m m).
4. Make 5 ml aliquots, and store at -20° C.

Alternate Protocol: Prepare desired amount of 100x SPF for use and add directly to medium without filtration, so the SPF is only filtered once with the medium.

3. ATV (0.5% Trypsin, 0.2g/L ETDA)*

Materials:

• Sodium Chloride, NaCl, M.W. 58.44
• Potassium Chloride, KCl, M.W. 74.56
• Sodium Bicarbonate, NaHCO₃, M.W. 84.01
• Dextrose, HOCH₂CH(CHOH)₄O, M.W. 180.16
• EDTA, M.W. 372.24
• Trypsin, Sigma Cat. #T-7409
• DDH₂O

Protocol for 1 Liter:

• Add to 500 ml of DDH₂O:
  • 8 g NaCl
  • 0.4 g KCl
  • 0.58 g NaHCO₃
  • 1 g Dextrose
  • 0.2 g EDTA
  • 0.5 g Trypsin
• Bring volume up to 1 L with DDH₂O, and pH to 7.4 with HCl.
• Incubate at 37° C for at least 1 hour to activate the trypsin.
• Sterile filter (0.22 m m) and make 10 ml aliquots.
• Refrigerate at 4° C overnight, and then store at -20° C.

* Commercially available products may be substituted.