Maraviroc mvc

When indicated, tumor-free mice received a hydrodynamic injection of Flt3L-encoding plasmid (FL) or empty vector. Hydrodynamic injection was performed by intravenous administration into the tail vein of 2 mL room temperature PBS containing 10 µg plasmid in less than 8 s. Where indicated, mice were inoculated with tumors 24 hours after the hydrodynamic injection. Intraperitoneal administration of 100 µg anti-DNGR-1-blocking antibodies (7H11, BioXcell) or isotype control (Sigma) was performed at days 5, 7, 9 and 12 after tumor inoculation. When indicated, 30 mg/kg maraviroc (MVC; Sigma) or vehicle were intraperitoneally administered concurrent with antibody administration. Cyclophosphamide (140 mg/kg; Sigma) was administered following a metronomic regime at days 8 and 14 of tumor development.

A time-of-addition study was conducted using a single-round replication assay. Briefly, TZM-bl cells were seeded in 96-well plates (2 × 10⁴ cells/well). After 24 h, SF162.LS pseudoviral particles were added to the cell monolayer. Maraviroc (MVC, Sigma-Aldrich, St. Louis, MO, USA) was used as a reference (control) drug. The tested compounds and control drug were then added to the cells at various points in time. Inhibition of pseudoviral infection was assessed after 48 h by measuring the level of luminescence, as above. Samples were tested in triplicates, and the experiment was repeated twice.

The HIV entry assay was performed by infecting 100,000 BAL CD4+ T cells, AMs or MDMs with 10⁵ infectious units of BLaM-Vpr HIV in a polypropylene FACS tube in a total volume of 100 μl after 1 h of pretreatment with DMSO or 40 μM maraviroc (MVC, Sigma). The cells were incubated at 37 °C for 12 h, then washed three times. Staining was performed by adding CCF2-AM along with Fixable Viability Dye eFluor 780 (eBioscience) to the cells and assaying for cleavage by flow cytometry^{98 (link)}.