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2 protocols using emtricitabine

1

Macrophage-Derived Foam Cell Formation

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PBMCs were thawed and monocytes were isolated using EasySep™ human monocytes isolation kit (STEMCELL Technologies Inc., Vancouver, BC, Canada), plated, and differentiated into macrophages for 8 days. M-CSF was used to stimulate MDM at a concentration of 50 ng/mL (STEMCELL Technologies Inc., Vancouver, BC, Canada). To induce foam cell formation, adherent macrophages were treated with 100 μg/mL oxLDL (Kalen Biomedical, Montgomery Village, MD, USA) for 24 h then double-stained with 0.4% oil red O for detection of lipids and anti-CD68 antibody for detection of macrophages. Double-stained CD68+/Oil Red O+-positive cells are considered foam cells. The percentage of macrophages that were also positive for oil red O was quantified in five 20× fields. Each point on the graph corresponds to the average value calculated from five different images captured for each sample. Antiretroviral drugs used were tenofovir (1 μg/mL), emtricitabine (2 μg/mL), and raltegravir (0.1 μg/mL) (Cayman chemical company, Ann Arbor, MI, USA). HIV tat, nef, and gp120 were used at a concentration of 100 ng/mL. Macrophage tropic HIV-ADA was used to infect MDMs at a concentration of 10 ng/mL of p24, a kind gift from Dr. Peter Gaskill (Drexel University School of Medicine, Philadelphia, PA, USA).
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2

Quantification of Tenofovir and Emtricitabine

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Triapine was obtained from Selleckchem (Houston, TX), tenofovir was generously donated by Dr. Lisa Rohan (University of Pittsburgh). Methimazole, N-benzyl imidazole, chlorogyline, deprenyl hydrochloride, (+) raloxifene hydrochloride, carnitine acetyltransferase, acetyl DL-carnitine, acetyl coA, triethanolamine and allopurinol were obtained from Sigma Aldrich (St Louis, MO). Human liver microsomes, human liver mitochondria, and human liver cytosol with high aldehyde oxidase and xanthine oxidase activity were obtained from Xenotech (Kansas City, KS). human liver cytosol was obtained from Corning (Corning, NY). Plated primary human hepatocytes were obtained from the Department of Surgery at the University of Pittsburgh. (Suppl.Table 1). Furafylline, phenyl piperidinyl propane, montelukast, sulfaphenazone, tranylcypromine hydrochloride, emtricitabine, disulfiram, and 4-methyl pyrazole were obtained from Cayman Chemical (Ann Arbor, MI). [2H6]-tenofovir and [2H3, 15N]-emtricitabine were purchased from Alsachim (Illkirch-Graffenstaden, France). 3-Benzyl nirvanol was purchased from Santa Cruz Biotechnology (Dallas, TX). Quinidine was obtained from Alfa Aesar (Tewksbury, MA). All samples generated were stored at −20 °C or lower until further analysis.
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