Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors at the NIH Blood Bank (Bethesda, MD). Monocytes were isolated by adherence and differentiated into macrophages (MDM) by culture in RPMI 1640 (Thermo Fisher; Waltham, MA) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic-antimycotic for ≥ 7 days. MDM were infected with HIVSF162 (>1 ng p24 per million cells) using polybrene (5 μg/ mL) (Sigma-Aldrich; St. Louis, MO) for four hours, then cells were washed with PBS and medium was replaced with complete RPMI containing dimethyl sulfoxide (DMSO) or darunavir (1 μM in DMSO). darunavir was obtained through the NIH AIDS Reagent Program (cat #11447), Division of AIDS, NIAID, NIH from Tibotec, Inc. Supernatants and cells were collected after seven days for product enhanced reverse transcriptase (PERT) assay [31 (link)] or Tat ELISA, respectively.
Protocol detail
Isolation and Infection of MDM with HIV
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Corresponding Organization : National Institutes of Health
Other organizations : Johns Hopkins Medicine, Johns Hopkins University, George Mason University, National Institute of Mental Health, University of California System, University of California, San Diego
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Variable analysis
independent variables
- Infection of macrophages (MDM) with HIV_SF162
- Reverse transcriptase activity (PERT assay)
- Tat expression (Tat ELISA)
- Macrophage (MDM) differentiation conditions (RPMI 1640 supplemented with 10% FBS and 1% antibiotic-antimycotic for ≥ 7 days)
- Polybrene concentration (5 μg/mL) used for HIV infection
- Darunavir concentration (1 μM) in DMSO
- DMSO treatment
- Not explicitly mentioned
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