368
Related Techniques
4. Dispense 100 fll culture medium to all wells, except wells A2-Al1.
5. Add 100 fll drug solution to each of the wells A2-All and B2-Bll (in
cases when all the wells are being used).
Dilution 1. After mixing the liquid in wells B2-Bll, transfer 100 fll of the liquid
in these wells to the corresponding wells C2-Cl1.
2. After mixing the liquid in wells C2-Cll, transfer 100 fll of the liquid
in these wells to the corresponding wells D2-D 11.
3. Repeat this dilution step up to the H wells.
4. Discard 100 fll of the liquid in each H well.
Cell 1. Harvest cells from a stock culture.
seeding 2. Centrifuge the cells at 150 g for 5 min.
3. Suspend the cells in the culture medium at a density of between 105
and 10 6 cells/ml.
4. Dispense 100 fll cell suspension to all the wells except for those labeled as negative controls. (Dispense the cell suspension into wells
labeled as positive controls first, then into wells containing the drug,
starting with wells with a lower concentration and finishing with wells
with a higher concentration.)
Maintenance 1. Culture the cells for 3-7 days.
2. At the end of the experimental culture, discard the culture medium.
Staining 1. Wash the cells with physiological solution.
Measurement
of optical
density
2. Fix the cells with methanol.
3. Stain the cells with 0.4% crystal violet solution.
1. Measure the optical density (OD) of each well at OD s9o using a
microplate reader.
2. Assign values of 100% and 0% to the mean OD of the positive and
negative controls, respectively, and then express the mean OD of each
concentration of the drug as a percentage.
Results
The data can be plotted on a graph with the log concentration of the
drug on the abscissa and the percentage OD on the ordinate. The concentration of 50 % inhibition (TCED so ) can be read directly from this
graph.
Comments
If the cells being used in the drug toxicity assay are not substrate dependent, apply the replicate culture method.
Related Techniques
4. Dispense 100 fll culture medium to all wells, except wells A2-Al1.
5. Add 100 fll drug solution to each of the wells A2-All and B2-Bll (in
cases when all the wells are being used).
Dilution 1. After mixing the liquid in wells B2-Bll, transfer 100 fll of the liquid
in these wells to the corresponding wells C2-Cl1.
2. After mixing the liquid in wells C2-Cll, transfer 100 fll of the liquid
in these wells to the corresponding wells D2-D 11.
3. Repeat this dilution step up to the H wells.
4. Discard 100 fll of the liquid in each H well.
Cell 1. Harvest cells from a stock culture.
seeding 2. Centrifuge the cells at 150 g for 5 min.
3. Suspend the cells in the culture medium at a density of between 105
and 10 6 cells/ml.
4. Dispense 100 fll cell suspension to all the wells except for those labeled as negative controls. (Dispense the cell suspension into wells
labeled as positive controls first, then into wells containing the drug,
starting with wells with a lower concentration and finishing with wells
with a higher concentration.)
Maintenance 1. Culture the cells for 3-7 days.
2. At the end of the experimental culture, discard the culture medium.
Staining 1. Wash the cells with physiological solution.
Measurement
of optical
density
2. Fix the cells with methanol.
3. Stain the cells with 0.4% crystal violet solution.
1. Measure the optical density (OD) of each well at OD s9o using a
microplate reader.
2. Assign values of 100% and 0% to the mean OD of the positive and
negative controls, respectively, and then express the mean OD of each
concentration of the drug as a percentage.
Results
The data can be plotted on a graph with the log concentration of the
drug on the abscissa and the percentage OD on the ordinate. The concentration of 50 % inhibition (TCED so ) can be read directly from this
graph.
Comments
If the cells being used in the drug toxicity assay are not substrate dependent, apply the replicate culture method.