Chapter 39
Examination of Drug Effects
Cultured invertebrate cells may be useful in drug toxicity tests, because
most invertebrate cell lines are easy to maintain. Unlike mammalian cells,
invertebrate cells can be cultured at lower and over a relatively wider
range of temperatures, and do not require a CO 2 incubator. Most insect
cell lines do not adhere to a substrate, and subculture is very easy. These
characteristics of invertebrate cell culture give some advantage to the
use of invertebrate cells over mammalian cells in drug toxicity tests.
Materials
- Centrifuge and centrifuge tubes
- Microplate reader
- Test tubes
- Pasteur pipettes
- 96-Well micro test plate
PROTOCOL
Equipment
- Culture medium
Solutions
- Drug solution (the drug being tested should be dissolved in culture
medium to a concentration twice that of the highest concentration to
be used in the test)
Procedure
1. Dissolve the drug to be tested directly in the culture medium if the
drug is water soluble. In the case of water-insoluble substances, dissolve the drug in an appropriate solvent. Ethanol or acetone are commonly used as solvents for water-insoluble drugs. When ethanol is to
be used as the solvent, prepare a solution of 2% Tween 80 in ethanol
(v/v), and then dissolve the drug in this solution.
2. If the addition of drug to the culture medium significantly alters the
pH of the medium, adjust the pH back to that of the drug-free medium with 1 N KOH and 1 N HCl. When a solvent is used, run the
experiment with solvent only and no drug as a control.
1. Mark wells 1A-1D and 12E-12H as positive controls.
2. Mark wells 1E-1H and 12A-12D as negative controls.
3. Use columns 2-11 for testing the drug. The experiment should be performed at least in duplicate. Using this set up, five drugs can be tested
simultaneously.
Incorporation
of drug
Assignment
of wells
Examination of Drug Effects
Cultured invertebrate cells may be useful in drug toxicity tests, because
most invertebrate cell lines are easy to maintain. Unlike mammalian cells,
invertebrate cells can be cultured at lower and over a relatively wider
range of temperatures, and do not require a CO 2 incubator. Most insect
cell lines do not adhere to a substrate, and subculture is very easy. These
characteristics of invertebrate cell culture give some advantage to the
use of invertebrate cells over mammalian cells in drug toxicity tests.
Materials
- Centrifuge and centrifuge tubes
- Microplate reader
- Test tubes
- Pasteur pipettes
- 96-Well micro test plate
PROTOCOL
Equipment
- Culture medium
Solutions
- Drug solution (the drug being tested should be dissolved in culture
medium to a concentration twice that of the highest concentration to
be used in the test)
Procedure
1. Dissolve the drug to be tested directly in the culture medium if the
drug is water soluble. In the case of water-insoluble substances, dissolve the drug in an appropriate solvent. Ethanol or acetone are commonly used as solvents for water-insoluble drugs. When ethanol is to
be used as the solvent, prepare a solution of 2% Tween 80 in ethanol
(v/v), and then dissolve the drug in this solution.
2. If the addition of drug to the culture medium significantly alters the
pH of the medium, adjust the pH back to that of the drug-free medium with 1 N KOH and 1 N HCl. When a solvent is used, run the
experiment with solvent only and no drug as a control.
1. Mark wells 1A-1D and 12E-12H as positive controls.
2. Mark wells 1E-1H and 12A-12D as negative controls.
3. Use columns 2-11 for testing the drug. The experiment should be performed at least in duplicate. Using this set up, five drugs can be tested
simultaneously.
Incorporation
of drug
Assignment
of wells