4 Notes
1. This protocol assumes that the testing condition is 7H9 broth
medium. However, it is flexible for users to make changes in
growth environment, strain, etc.
2. Drugs can either be dispensed manually (e.g., through serial
dilution) or with a digital dispenser (such as the HP D300E
Digital Dispenser). A digital dispenser allows the experiment to
be done more efficiently and with fewer plate effects if the
randomization function is used.
3. The volume of culture must be adjusted depending on the plate
used in the experiment. Both 96- and 384-well plate can be
used for this assay depending on experimental needs. 384-well
is more efficient for large-scale testing.
4. For assays that last longer than 5 days using 384-well plates, it
is necessary to ensure the liquid does not evaporate before the
end of the assay. An ad hoc humidifier can be made by adding
cotton balls or paper towel soaked with water into open 50 mL
conical tubes within the same zip-loc bags as the assay plates.
5. The reading time and measurement type can be adjusted for
different experimental purposes or growth conditions.
6. Dose spacing and the resolution of the dose sampling can be
changed depending on the nature of the compounds (e.g., for
different potencies). However, as mentioned in the protocol, it
is important to keep the dose spacing consistent among all
compounds used in one experiment.
7. For higher-order combinations, the combination dose sampling will combine the single-drug dose sampling array at
dose levels reduced by a factor of n (where n is the number of
drugs in combination). For example, the 7th dose of a ten-dose
resolution dose sampling in a 3-drug combination (drugs A, B,
and C) will be a combination of a third of the amount of each
drug used in the 7th dose of the single-drug dose spacing.
8. Wells for Z-factor calculation and blank background can be
added to the assay for quality control assessment. A Z-factor
is obtained by calculating mean and standard deviation of
positive control (samples treated with MIC or higher levels of
drug) and negative control (untreated wells),
Z À f actor ¼
1 À 3ðσ p þ σ n Þ
jμ p À μ n j
,
where σ p and σ n are standard deviation of positive and negative
control, respectively; μ p and μ n are mean of positive and negative control, respectively. A Z-factor of 0.5 or above is typically
Efficient Measurement of Drug Interactions with DiaMOND (Diagonal. . .
711
1. This protocol assumes that the testing condition is 7H9 broth
medium. However, it is flexible for users to make changes in
growth environment, strain, etc.
2. Drugs can either be dispensed manually (e.g., through serial
dilution) or with a digital dispenser (such as the HP D300E
Digital Dispenser). A digital dispenser allows the experiment to
be done more efficiently and with fewer plate effects if the
randomization function is used.
3. The volume of culture must be adjusted depending on the plate
used in the experiment. Both 96- and 384-well plate can be
used for this assay depending on experimental needs. 384-well
is more efficient for large-scale testing.
4. For assays that last longer than 5 days using 384-well plates, it
is necessary to ensure the liquid does not evaporate before the
end of the assay. An ad hoc humidifier can be made by adding
cotton balls or paper towel soaked with water into open 50 mL
conical tubes within the same zip-loc bags as the assay plates.
5. The reading time and measurement type can be adjusted for
different experimental purposes or growth conditions.
6. Dose spacing and the resolution of the dose sampling can be
changed depending on the nature of the compounds (e.g., for
different potencies). However, as mentioned in the protocol, it
is important to keep the dose spacing consistent among all
compounds used in one experiment.
7. For higher-order combinations, the combination dose sampling will combine the single-drug dose sampling array at
dose levels reduced by a factor of n (where n is the number of
drugs in combination). For example, the 7th dose of a ten-dose
resolution dose sampling in a 3-drug combination (drugs A, B,
and C) will be a combination of a third of the amount of each
drug used in the 7th dose of the single-drug dose spacing.
8. Wells for Z-factor calculation and blank background can be
added to the assay for quality control assessment. A Z-factor
is obtained by calculating mean and standard deviation of
positive control (samples treated with MIC or higher levels of
drug) and negative control (untreated wells),
Z À f actor ¼
1 À 3ðσ p þ σ n Þ
jμ p À μ n j
,
where σ p and σ n are standard deviation of positive and negative
control, respectively; μ p and μ n are mean of positive and negative control, respectively. A Z-factor of 0.5 or above is typically
Efficient Measurement of Drug Interactions with DiaMOND (Diagonal. . .
711