How to prepare materials for growth media

Materials for Cell Growth Medium

Ham’s F-12

Source: Hyclone SH30026FS from Fisher Scientific

Fungizone

Source: Amphotericin B from Fisher Scientific, Catalog #BP2645-50

Gentamicin Sulfate

Source: Gemini Bioproducts Catalog # 400-108

Insulin

Source: Gemini Bioproducts #700-112P

Preparation:

  1. Empty 100 mg vial of bovine pancreas insulin into a 200 ml beaker.
  2. Wash vial with 1.0 ml 0.1 N HCl. Pour wash into insulin and swirl to dissolve. Make sure insulin is dissolved before proceeding. If necessary, add more HCl.
  3. Add 99 ml DDH2O to the beaker.
  4. Filter sterilize through a 0.22 micron filter.
  5. Aliquot 20 ml volumes into 50 ml tubes, store at -20°C.
  6. After thawing, store aliquots at 4°C.

Hydrocortisone

Source: Sigma Catalog #H-4001

Preparation:

  1. Place 40 mg hydrocortisone in a sterile 50 mL tube.
  2. Add 20 mL EtOH.
  3. Add 20 ml sterile DDH2O
  4. Do NOT filter.
  5. Divide into 5 mL aliquots, store at -20°C.

Fetal Bovine Serum

Source: Atlanta Biologicals #S11050; Gemini Bioproducts 900-108

Store 500 mL bottles at -20°C. When thawed, aliquot FBS into 50 mL tubes and store at 4°C.

Bovine Serum Albumin

Source: Millipore Catalog #126579

Preparation:

  1. Add 10 g BSA to 500 mL Hank’s F-12 medium.
  2. Stir at RT until completely dissolved.
  3. Filter with 0.22 micron filter.
  4. Store at 4°C.

Ethanolamine

Source: Sigma Catalog #E0135

Store at room temperature.

HEPES

Source: Sigma Catalog #H3375

Preparation:

  1. Add 23.83 g HEPES to 100 ml distilled water.
  2. Stir until dissolved, filter with 0.22 micron filter.
  3. Store at 4°C.

Transferrin

Source: Sigma Catalog #T2252

Preparation:

  1. Dissolve 100 mg transferrin in 40 mL DDH2O
  2. Filter with 0.22 micron filter
  3. Store at -20°C in 1 mL aliquots.
  4. Can be stored at 4°C after thawing.

Triiodo thyronine (T3)

Source: Sigma Catalog #T5516

Preparation:

  1. Dissolve 1.0mg Triiodo Thyronine in 1 mL 0.01N NaOH.
  2. Pipette resulting solution into 50 mL tube and add 49 mL 1x Hank’s Buffered Salt Solution.
  3. Filter-sterilize using a 0.22 micron filter.
  4. Aliquot into 1 mL volumes and store at -20°C.
  5. Can be stored at 4°C after thawing.

Sodium Selenite (Se)

Source: Sigma Catalog #S9133

Preparation:

  1. Dissolve 1 mg in 50 mL Hank’s Buffered Salt Solution.
  2. Filter-sterilize using a 0.22 micron filter.
  3. Aliquot into 1 mL volumes and store at -20°C.
  4. Can be stored at 4°C after thawing.

Epidermal Growth Factor

Source: Sigma Catalog #E9644

Preparation:

  1. Resuspend 0.2mg EGF in 20 mL sterile DDH2O
  2. Do NOT filter.
  3. Aliquot 500 μL into 1 mL sterile tubes.
  4. Store at -20°C.

Prolactin

Source: Sigma Catalog #L6520, 250 IU

Preparation:

  1. Calculate amount necessary to make stock of 1 mg/ml (based on IU/mg).
  2. Follow manufacturer’s recommended procedure for dissolving and sterilizing.
  3. Store in aliquots at -20°C.

Lysophosphatidic Acid (LPA)

Source: Sigma Catalog #L7260, 25 mg

Preparation:

  1. Dissolve entire contents (25 mg) into 5.7 mg calcium and magnesium free 1x HGSS to achieve a stock concentration of 10 mM.
  2. Sterile Filter using low protein binding filter and store in 0.5 mL aliquots at -20°C.

Β-Estradiol

Source: Sigma Catalog #E2257, 1 mg

Preparation:

  1. Dissolve 1 mg powder in 1 mL absolute ethanol while gently swirling, then add 49 mL sterile Ham’s F-12.
  2. Store at -20°C in 1 mL aliquots.

Progesterone

Source: Sigma P7556, 1 mg

Preparation:

  1. Dissolve 1 mg powder in 50 mL sterile Ham’s F-12.
  2. Store at -20°C in 1 mL aliquots.

Cholera Toxin

Source: Sigma Catalog # C3012, 1 mg

Preparation:

  1. Dissolve in 10 mL HBSS.
  2. Sterile filter with 0.22 micron filter.
  3. Store at -20°C in 1 mL aliquots.

Back Home