dose of drug A and half dose of drug B. Design the dose
sampling for a pairwise combination so that the nth position
of each combination and single dose sampling should have the
same total number of dose units (Fig. 4). For high-order
combinations (e.g., three or more drugs), see Note 7.
4. In a 384-well plate, reserve 28 wells for untreated (negative
control) and standard wells (wells treated with the most potent
compounds at MIC level, serving as positive control) for
Z-factor calculation (see Note 8).
5. Do not use edge wells (i.e., the first and last columns, and first
and last rows) for dose responses because plate effects are more
pronounced around the edges of plates.
6. Reserve several wells for medium-only and use for background
reading subtraction during analysis.
3.4 Drug Interaction
Experiment
1. Dispense drugs manually (or with an automatic dispenser)
based on the design in Subheading 3.3.
2. Add culture (from Subheading 3.1) and collect OD 600 data
after 5 days.
3.5 Data Analysis
1. Subtract background readings from sample reads (RawOD).
The background is calculated as the median of all medium-only
containing wells to give the background-subtracted response,
C:
C ¼ RawOD À median m
ð Þ,
where m is the medium wells that neither contain bacteria nor
drugs.
Fig. 4 Schematic of combination drug dose spacing and relationship between single drug dose spacing and
the equipotent dose spacing combination. The dose units must be equivalent for every dose spacing design in
the DiaMOND assay, and this is achieved by combining half of the dose units for each of drug A and drug B into
the corresponding dose position for the pairwise combination
708
Nhi Van et al.
sampling for a pairwise combination so that the nth position
of each combination and single dose sampling should have the
same total number of dose units (Fig. 4). For high-order
combinations (e.g., three or more drugs), see Note 7.
4. In a 384-well plate, reserve 28 wells for untreated (negative
control) and standard wells (wells treated with the most potent
compounds at MIC level, serving as positive control) for
Z-factor calculation (see Note 8).
5. Do not use edge wells (i.e., the first and last columns, and first
and last rows) for dose responses because plate effects are more
pronounced around the edges of plates.
6. Reserve several wells for medium-only and use for background
reading subtraction during analysis.
3.4 Drug Interaction
Experiment
1. Dispense drugs manually (or with an automatic dispenser)
based on the design in Subheading 3.3.
2. Add culture (from Subheading 3.1) and collect OD 600 data
after 5 days.
3.5 Data Analysis
1. Subtract background readings from sample reads (RawOD).
The background is calculated as the median of all medium-only
containing wells to give the background-subtracted response,
C:
C ¼ RawOD À median m
ð Þ,
where m is the medium wells that neither contain bacteria nor
drugs.
Fig. 4 Schematic of combination drug dose spacing and relationship between single drug dose spacing and
the equipotent dose spacing combination. The dose units must be equivalent for every dose spacing design in
the DiaMOND assay, and this is achieved by combining half of the dose units for each of drug A and drug B into
the corresponding dose position for the pairwise combination
708
Nhi Van et al.