Primary monocytes at 1 x 10⁵ cells were seeded in a flask and supplemented with RPMI 1640 + GM-CSF + IL-4 and incubated at 37°C for 6 days for monocyte to macrophage transformation.

On day 7, monocyte-derived macrophages [MDM] were harvested, and 1 x 10⁵ cells per well were plated. Fentanyl at 10 ug/mL was added and incubated for 24 hours. After 24 hours, cells were infected with HIV_YK-JRCSF and incubated for 2 hours. The cells were rinsed three times to remove any unbound virus and replaced with fresh media with fentanyl and incubated for 3 days. Cells were harvested at the end of 72 hours and total RNA extraction was performed. Genes of interest were quantified by brilliant III ultrafast SYBR qRT-PCR. Error bars represent the standard deviations between replicates. Data were normalized to beta-globin expression and fold-change in expression was calculated by the 2 ^−ΔΔCT method. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (PDF)