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General Methods
Place approximately 20 ml of 1 N HCl in a 1,000-ml beaker.
Dissolve 125 mg L-cystine and 250 mg L-tyrosine in the 1 N HCl.
Add approximately 900 ml distilled water to the solution
Stir the solution on a magnetic stirrer.
Dissolve the following in the solution, one after the other: 1,125 mg La-alanine, 1,000 mg ~-alanine, 3,500 mg L-arginine hydrochloride,
1,750 mg L-aspartic acid, 1,750 mg L-asparagine, 3,000 mg L-glutamic
acid, 3,000 mg L-glutamine, 3,250 mg glycine, 12,500 mg L-histidine,
250 mg L-isoleucine, 375 mg L-leucine, 3,125 mg L-lysine hydrochloride, 250 mg L-methionine, 1,750 mg L-proline, 750 mg L-phenylalanine, 2,750 mg L-serine, 875 mg L-threonine, 500 mg L-tryptophan,
500 mg L-valine, 133.4 g sucrose, 2.0 g fructose, 3.5 g glucose, 3,350 mg
L-malic acid, 300 mg succinic acid, 275 mg fumaric acid, and 1,850 mg
a-ketoglutaric acid.
Add distilled water to make up a final volume of 1,000 ml.
Store the solution at -20°C (can be kept for several years).
- GMA vitamin stock solution I (lOOO-fold concentrated)
Warm 100 ml distilled water to 50°C,
Dissolve 2 mg riboflavin in the distilled water.
When the solution becomes clear, let it cool to room temperature.
Dissolve 2 mg thiamine hydrochloride, 2 mg calcium pantothenate,
2 mgp-aminobenzoic acid, 2 mg inositol, 2 mg pyridoxine hydrochloride, 2 mg niacin, and 20 mg choline chloride in the solution.
Store the solution at -20°C (can be kept for several years).
- GMA vitamin stock solution II (lOOO-fold concentrated)
Take 100 ml distilled water.
Adjust the pH of the water up to 10.0 with KOH.
Dissolve 2 mg folic acid and 1 mg biotin in the solution.
When the folic acid and biotin have dissolved completely, adjust the
pH of the solution to 7.0 with 1 N HCl.
Store the solution at -20°C (can be kept for several years).
Sera - FBS
- Insect hemolymph, heat treated (60°C, 30 min)
Procedure
1. To prepare 1,000 ml of the ready-to-use GMA, mix 100 ml GMA Salt
Stock Solution A, 100 ml GMA Salt Stock Solution B, 200 ml GMA Stock
Solution OSA, 1 ml GMA Vitamin Stock Solution I, and 1 ml GMA
Vitamin Stock Solution II. Dissolve 30 mg penicillin G sodium salt
and 100 mg streptomycin sulfate in this mixture. Then, add distilled
water to 1,000 ml.
2. Adjust the pH of the solution to 6.3 with concentrated KOH.
3. After sterilization of the solution by passing it through a membrane
filter of 0.2 11m pore size, add one-tenth the volume of FBS or 3%-5%
sterilized heat-treated insect hemolymph to the medium.
General Methods
Place approximately 20 ml of 1 N HCl in a 1,000-ml beaker.
Dissolve 125 mg L-cystine and 250 mg L-tyrosine in the 1 N HCl.
Add approximately 900 ml distilled water to the solution
Stir the solution on a magnetic stirrer.
Dissolve the following in the solution, one after the other: 1,125 mg La-alanine, 1,000 mg ~-alanine, 3,500 mg L-arginine hydrochloride,
1,750 mg L-aspartic acid, 1,750 mg L-asparagine, 3,000 mg L-glutamic
acid, 3,000 mg L-glutamine, 3,250 mg glycine, 12,500 mg L-histidine,
250 mg L-isoleucine, 375 mg L-leucine, 3,125 mg L-lysine hydrochloride, 250 mg L-methionine, 1,750 mg L-proline, 750 mg L-phenylalanine, 2,750 mg L-serine, 875 mg L-threonine, 500 mg L-tryptophan,
500 mg L-valine, 133.4 g sucrose, 2.0 g fructose, 3.5 g glucose, 3,350 mg
L-malic acid, 300 mg succinic acid, 275 mg fumaric acid, and 1,850 mg
a-ketoglutaric acid.
Add distilled water to make up a final volume of 1,000 ml.
Store the solution at -20°C (can be kept for several years).
- GMA vitamin stock solution I (lOOO-fold concentrated)
Warm 100 ml distilled water to 50°C,
Dissolve 2 mg riboflavin in the distilled water.
When the solution becomes clear, let it cool to room temperature.
Dissolve 2 mg thiamine hydrochloride, 2 mg calcium pantothenate,
2 mgp-aminobenzoic acid, 2 mg inositol, 2 mg pyridoxine hydrochloride, 2 mg niacin, and 20 mg choline chloride in the solution.
Store the solution at -20°C (can be kept for several years).
- GMA vitamin stock solution II (lOOO-fold concentrated)
Take 100 ml distilled water.
Adjust the pH of the water up to 10.0 with KOH.
Dissolve 2 mg folic acid and 1 mg biotin in the solution.
When the folic acid and biotin have dissolved completely, adjust the
pH of the solution to 7.0 with 1 N HCl.
Store the solution at -20°C (can be kept for several years).
Sera - FBS
- Insect hemolymph, heat treated (60°C, 30 min)
Procedure
1. To prepare 1,000 ml of the ready-to-use GMA, mix 100 ml GMA Salt
Stock Solution A, 100 ml GMA Salt Stock Solution B, 200 ml GMA Stock
Solution OSA, 1 ml GMA Vitamin Stock Solution I, and 1 ml GMA
Vitamin Stock Solution II. Dissolve 30 mg penicillin G sodium salt
and 100 mg streptomycin sulfate in this mixture. Then, add distilled
water to 1,000 ml.
2. Adjust the pH of the solution to 6.3 with concentrated KOH.
3. After sterilization of the solution by passing it through a membrane
filter of 0.2 11m pore size, add one-tenth the volume of FBS or 3%-5%
sterilized heat-treated insect hemolymph to the medium.