burden of combination testing. For example, DiaMOND enables
us to determine a 3-way drug interaction with just 4% of the
measurements required from a checkerboard assay.
DiaMOND is an experimental design and analysis methodology; therefore, it has the flexibility to be adapted to a wide variety of
growth conditions and disease models. Though DiaMOND experiments are simple to perform, they require care and precision to
obtain consistent results. Pitfalls include inconsistency in the bacterial growth stage or age of growth medium, repeated freeze-thaw
cycles of drug aliquots, and nonoptimized dose spacing. The steps
in this protocol are designed to avoid these pitfalls.
1.1 DiaMOND
Overview
DiaMOND experiments are performed in two major steps. The
purpose of the first step is to determine the coarse shape of the
single-drug dose response curves so that combination dose
responses for the subsequent DiaMOND experiments are equipotent and that the dynamic range of combination treatment is
achieved. In this first step, we obtain an initial dose response
curve for single drugs from which we calculate different growth
inhibition concentrations (such as the IC 50 and IC 90 ) to design an
optimized drug interaction experimental layout. The second step is
the DiaMOND combination experiment where we measure both
single- and combination-dose responses using an optimized dose
spacing and range from the first step. To determine drug interactions, we compare the observed inhibitory concentrations from the
combination dose responses to the expected inhibitory concentrations based on calculations from the single drug dose responses.
The DiaMOND workflow is summarized in Fig. 2.
2 Materials
2.1 Culturing
Bacteria
1. Mycobacterium tuberculosis such as Erdman or H37Rv, etc.
2. 30 mL air-tight inkwells.
3. 37
C shaking incubator (100 rpm).
4. 7H9 broth medium: To make 1000 mL, add 4.7 g of Middlebrook 7H9 powder, 2.5 mL of 20% v/v Tween80, 4 mL of
50% v/v Glycerol, and 100 mL of OADC supplement (Oleic
Acid-Albumin-Dextrose-Catalase enrichment) to 900 mL of
deionized water, and filter sterilize through 0.2 μM filter.
5. Microplate absorbance reader (e.g., Biotek Synergy Neo2
Hybrid Multi-Mode Reader).
2.2 Dose Centering
and DiaMOND Assay
1. Antibiotics of interest.
2. DMSO and other solvents (drug-specific).
Efficient Measurement of Drug Interactions with DiaMOND (Diagonal. . .
705
us to determine a 3-way drug interaction with just 4% of the
measurements required from a checkerboard assay.
DiaMOND is an experimental design and analysis methodology; therefore, it has the flexibility to be adapted to a wide variety of
growth conditions and disease models. Though DiaMOND experiments are simple to perform, they require care and precision to
obtain consistent results. Pitfalls include inconsistency in the bacterial growth stage or age of growth medium, repeated freeze-thaw
cycles of drug aliquots, and nonoptimized dose spacing. The steps
in this protocol are designed to avoid these pitfalls.
1.1 DiaMOND
Overview
DiaMOND experiments are performed in two major steps. The
purpose of the first step is to determine the coarse shape of the
single-drug dose response curves so that combination dose
responses for the subsequent DiaMOND experiments are equipotent and that the dynamic range of combination treatment is
achieved. In this first step, we obtain an initial dose response
curve for single drugs from which we calculate different growth
inhibition concentrations (such as the IC 50 and IC 90 ) to design an
optimized drug interaction experimental layout. The second step is
the DiaMOND combination experiment where we measure both
single- and combination-dose responses using an optimized dose
spacing and range from the first step. To determine drug interactions, we compare the observed inhibitory concentrations from the
combination dose responses to the expected inhibitory concentrations based on calculations from the single drug dose responses.
The DiaMOND workflow is summarized in Fig. 2.
2 Materials
2.1 Culturing
Bacteria
1. Mycobacterium tuberculosis such as Erdman or H37Rv, etc.
2. 30 mL air-tight inkwells.
3. 37
C shaking incubator (100 rpm).
4. 7H9 broth medium: To make 1000 mL, add 4.7 g of Middlebrook 7H9 powder, 2.5 mL of 20% v/v Tween80, 4 mL of
50% v/v Glycerol, and 100 mL of OADC supplement (Oleic
Acid-Albumin-Dextrose-Catalase enrichment) to 900 mL of
deionized water, and filter sterilize through 0.2 μM filter.
5. Microplate absorbance reader (e.g., Biotek Synergy Neo2
Hybrid Multi-Mode Reader).
2.2 Dose Centering
and DiaMOND Assay
1. Antibiotics of interest.
2. DMSO and other solvents (drug-specific).
Efficient Measurement of Drug Interactions with DiaMOND (Diagonal. . .
705