Tween 80

Lumefantrine (AK Scientific, Union City, CA), chloroquine (Sigma-Aldrich, St. Louis, MO), pyrimethamine (AK Scientific), atovaquone (Sigma-Aldrich), and ferroquine (Medicines for Malaria Venture) were formulated in 1% methylcellulose (Acros Organics, Morris Plains, NJ) and 0.1% Tween 80 (Thermo Fisher Scientific, Fair Lawn, NJ) in double-distilled water. Piperaquine (AK Scientific) and artefenomel (Medicines for Malaria Venture) were formulated in 0.1% hydroxypropyl-methylcellulose (Sigma-Aldrich), 0.5% benzyl alcohol (Sigma-Aldrich), 0.4% Tween 80, 0.9% sodium chloride (Fisher Scientific, Loughborough, LE, UK) in double-distilled water. In some studies, Piperaquine was formulated in double-distilled water. Pyronaridine (Medicines for Malaria Venture) was formulated in 0.5% hydroxypropyl-methylcellulose and 0.1% Tween 80.
Before drug administration, each infected mouse was randomly assigned to its corresponding treatment. Drug treatment started when infected mice had ~1.4% patent parasitemia in peripheral blood (day 1 of study, designated P₀). The treatments were administered by oral gavage with 20-gauge straight reusable feeding needles (Fine Science Tools GmbH, Heidelberg, Germany). The volume of administration for the per os (p.o.) route was 10 mL/kg of body weight unless otherwise stated.

Mtb H37Rv strain was grown in 7H9 broth (Sigma-Aldrich) supplemented with 0.05% Tween 80 (Thermo Fisher) and 10% oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) at 37 °C. H37Rv expressing the red fluorescent protein (H37Rv-RFP) was a kind gift from Dr Joel Ernst (University of California San Francisco, USA) and grown in 7H9 broth (BD Biosciences) supplemented with 0.05% Tween 80 (Thermo Fisher), 10% OADC and 30 μg ml⁻¹ kanamycin (Sigma-Aldrich) at 37 °C. Bacteria in mid-log phase (optical density (OD) of 0.6–1.0) were centrifuged at 5,000 r.p.m. for 10 min, resuspended in fresh 7H9 media and frozen at −80 °C in aliquots of ~10⁸ bacilli per ml.

Mtb H37Rv strain was grown in 7H9 broth (Sigma-Aldrich) supplemented with 0.05% Tween 80 (Thermo Fisher Scientific) and 10% oleic acid–albumin–dextrose–catalase (OADC; BD Biosciences) at 37°C. Mtb H37Rv expressing the red fluorescent protein (H37Rv-RFP) was a kind gift from Dr. Joel Ernst (University of California San Francisco, San Francisco, CA) and grown in 7H9 broth (BD Biosciences) supplemented with 0.05% Tween 80 (Thermo Fisher Scientific), 10% OADC, and 30 μg/ml kanamycin (Sigma-Aldrich) at 37°C. Bacteria in mid-log phase (OD 0.6-1.0) were centrifuged at 5,000 rpm for 10 min, resuspended in fresh 7H9 media, and frozen at −80°C in aliquots of ∼10⁸ bacilli/ml.