from previous studies, we recommend an initial dose centering
experiment using serial twofold dilution with ten doses where the
reported MIC is situated at least fivefold below the maximum
concentration used. If the MIC is unknown, a larger number of
doses can be used. If inhibition is highly variable or the full dose
response curve is not reached, repeat Subheading 3.2 with a dose
modification as appropriate.
1. Dispense antibacterial compounds into wells either manually or
with digital dispenser (see Note 2).
2. Dispense 50 μL of culture (from step 4 in Subheading 3.1) to
each well of a 384-well plate (see Note 3).
3. Place lid on 384-well plate and incubate standing at 37
C.
Keep the plates humidified to prevent dehydration (see
Note 4).
4. Read OD 600 after 5 days (see Note 5).
5. Calculate IC 50 and IC 90 . This step can be done by plotting
growth inhibition (OD 600 ) and manually extrapolating the
IC 50 and IC 90 .
3.3 Drug Interaction
Experimental Design
The experimental design shown in Fig. 3 is meant as a default or
starting point that can be adapted based on the experimental needs.
Here, a ten-dose resolution is used for all dose responses (see
Note 6).
1. Design dose ranges and spacing based on single-drug dose
centering (from Subheading 3.2).
2. Center the IC levels at an equipotent unit for all testing drugs
(e.g., IC 90 for all the drugs is centered at approximately the 7th
(out of 10) doses).
3. Dose sampling is composed of half of each of the single drug,
e.g., pairwise combination of drugs A and B will require half
Fig. 3 Schematic of single drug dose spacing and relationship between drug concentration (mM) and dose unit
(unitless). The dose units must be equivalent for every dose response in the DiaMOND assay
Efficient Measurement of Drug Interactions with DiaMOND (Diagonal. . .
707
experiment using serial twofold dilution with ten doses where the
reported MIC is situated at least fivefold below the maximum
concentration used. If the MIC is unknown, a larger number of
doses can be used. If inhibition is highly variable or the full dose
response curve is not reached, repeat Subheading 3.2 with a dose
modification as appropriate.
1. Dispense antibacterial compounds into wells either manually or
with digital dispenser (see Note 2).
2. Dispense 50 μL of culture (from step 4 in Subheading 3.1) to
each well of a 384-well plate (see Note 3).
3. Place lid on 384-well plate and incubate standing at 37
C.
Keep the plates humidified to prevent dehydration (see
Note 4).
4. Read OD 600 after 5 days (see Note 5).
5. Calculate IC 50 and IC 90 . This step can be done by plotting
growth inhibition (OD 600 ) and manually extrapolating the
IC 50 and IC 90 .
3.3 Drug Interaction
Experimental Design
The experimental design shown in Fig. 3 is meant as a default or
starting point that can be adapted based on the experimental needs.
Here, a ten-dose resolution is used for all dose responses (see
Note 6).
1. Design dose ranges and spacing based on single-drug dose
centering (from Subheading 3.2).
2. Center the IC levels at an equipotent unit for all testing drugs
(e.g., IC 90 for all the drugs is centered at approximately the 7th
(out of 10) doses).
3. Dose sampling is composed of half of each of the single drug,
e.g., pairwise combination of drugs A and B will require half
Fig. 3 Schematic of single drug dose spacing and relationship between drug concentration (mM) and dose unit
(unitless). The dose units must be equivalent for every dose response in the DiaMOND assay
Efficient Measurement of Drug Interactions with DiaMOND (Diagonal. . .
707