For ease of use, prepare aliquots of all solutions and keep frozen. Aliquots may be placed in a Styrofoam rack and the rack labeled with stock number, solution name, aliquot volume, and date.
The choice of water for making stocks depends upon local availability of highly-purified water. We make up stock solutions using Tissue Culture Water purchased from Sigma. For all other media (OCM, saline, etc.), we use Millique water or distilled and deionized water.
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1 2 3 4 5 6 7 8 8A 9 9A 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Stock 1:Na lactate. Purchase as a 98% syrup. Follow manufacturer’s indications for expiration date. Store at 4°C.
Stock 2: Na pyruvate. Dissolve 0.220 g sodium pyruvate in 100 ml water. Sterile-filter into an aluminum-foil wrapped 100 ml bottle and store at 4°C for 1 mo.
Stock 3: Bovine Steer Serum (BSS). Prepare 10 ml aliquots of BSS in sterile 13 ml tubes and store at -20°C indefinitely.
Stock 4: BSS/Hep. Add 1000 USP units of sterile heparin (dissolved in 3-5 ml of water and sterilize through a syringe filter) into 500 ml BSS (Stock 3). Store in 8 ml aliquots in sterile 13 ml tubes indefinitely at -20°C.
Stock 5: Estradiol. Dissolve 1 to 3 mg estradiol in ethanol for a final concentration of 1 mg/ml. Store in a glass container at -20°C for up to 2 months.
Stock 6: Folltropin. Reconstitute Folltropin-V as directed by manufacturer to prepare a 20 mg/ml solution. Place 150 ml aliquots into sterile 1.5 ml microcentrifuge tubes and store indefinitely at -20°C.
Stock
7: Heparin. Dissolve 20 mg in 10
ml water. Pipet 300 ml aliquots and store at -20°C in 1.5 ml
microcentrifuge tubes indefinitely.
Stock 8: Gentamicin. Dilute to 5 mg/ml concentration with water and sterile filter. Pipet 1 ml aliquots into sterile 4 ml tubes and store at -20°C indefinitely.
Stock 8A: Gentamicin. When preparing Stock 8, prepare a few extra tubes of 10 ml aliquots in sterile microcentrifuge tubes and store at -20°C indefinitely.
Stock 9: PHE Mix. Prepare as fresh solutions primary stocks of 1 mM hypotaurine (1.09 mg in 10 ml saline), 2 mM penicillamine (3 mg in 10 ml saline) and 250 mM epinephrine [dissolve 1.83 mg in 40 ml of a lactate-metabisulfite solution (stock 9a)]. Epinephrine is easily oxidized by direct light so take precautions to avoid this problem (wrap in aluminum foil or place in dark container). Combine 10 ml of 1 mM hypotaurine, 10 ml of 2 mM penicillamine, 4 ml of 250 mM epinephrine and 16 ml of saline and sterile filter. Aliquot 400 ml of PHE Mix into sterile 1.5 ml microcentrifuge tubes and store in a light resistant container at -20°C indefinitely. Upon retrieval of PHE mix for use, wrap tube in aluminum foil.
Stock 9a: Lactate-metabisulfite solution. Add 77 ml of a 98% Na lactate syrup (or the equivalent volume if a lower percent lactate syrup is used) and 50 mg Na metabisulfite to 50 ml water. Make fresh for each use.
Stock 10: Glutamine (1ml). Prepare stock solution of 1.5 g glutamine/100 ml water, sterile filter and make 1 ml aliquots in sterile 4 ml tubes and store at -20°C indefinitely.
Stock 11: Glutamine (4 ml). Prepare stock solution of 1.5 g glutamine/100 ml water, sterile filter and store 4 ml aliquots in 13 ml tubes at -20°C indefinitely.
Stock 12: MgCl2 for Percoll. Prepare 0.1 M stock by adding 0.203 g MgCl2 to 10 ml water. Sterile filter and store at 4°C indefinitely.
Stock 13: CaCl2 for Percoll. Prepare 1 M stock by adding 0.735 g CaCl2+2H2O to 5 ml water. Sterile filter and store at 4°C indefinitely.
Stock 14: Hyaluronidase. Prepare stock solution of type IV hyaluronidase at 10,000 units/ml in saline, sterilize through a 0.2 mm filter into a sterile tube, and prepare 100 ml aliquots in sterile microcentrifuge tubes and store at -20°C indefinitely.
Stock 15: Pen/Strep (4 ml). Thaw 100 ml bottle of pen/strep and aliquot 4 ml into sterile 5 ml tubes and store at -20°C indefinetely.
Stock 16: Pen/Strep (10 ml). Thaw 100 ml bottle of pen/strep and aliquot 10 ml into sterile 13 ml tubes and store at -20°C indefinetely.
Stocks 17-23 (prepare only if you
make your own TL media)
Stock 17: NaCl. Dissolve 6.665 g in 50 mL water. Sterile filter and store at 4°C.
Stock 18: KCl. Dissolve 0.588 g in 50 mL water. Sterile filter and store at 4°C.
Stock 19: NaHCO3. Dissolve 1.052 g in 50 mL water. Sterile filter and store at 4°C for one week only.
Stock 20: PO4. Dissolve 0.235 g NaH2PO4+H2O in 50 mL water. Sterile filter and store at 4°C.
Stock 21: 1 M HEPES. Add 119 g of HEPES to 400 mL water. Adjust pH to 7.0 and bring volume up to 500 mL. Sterile filter and cover container with aluminum foil; store at 4°C indefinitely.
Stock 22: CaCl2 for TL. Dissolve 1.470 g CaCl2+2H2O in 50 mL water. Sterile filter and store at 4°C.
Stock 23: MgCl2 for TL. Dissolve 1.017 g MgCl2+ 6H2O in 50 mL water. Sterile filter/store at 4°C.
Stock 24: Fetal calf serum. Prepare 100 ml aliquots of heat-inactivated fetal calf serum in sterile microcentrifuge tubes and store at -20°C
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