Cobas ampliprep system v2

Humanized CD34⁺-NSG mice were generated as reported earlier^{31 (link)}. 20–22 week old mice were administered SAG (20 μg/g body weight, Cayman Chemicals, USA) or vehicle control (PEG-400 in PBS, Sigma Aldrich, USA) intraperitoneally (i.p.). The mice were injected i.p. with HIV-infected PHA blasts (2 × 10⁶ cells/mouse) 18–20 hours later. 24 hours post-infection, the mice were given another dose of SAG and were sacrificed on the next day (day 4), or on day 35. Whole blood was collected by cardiac exsanguination. The mice were then perfused and brains were harvested. Blood was either used to isolate plasma or to perform flow cytometry to estimate cell counts for CD4+ T cells and p24 expression as described above (N = 3 per group). Plasma was used to perform S100B ELISA (as per manufacturer’s instructions, #SEA567Mu, Clone-Cloud Corp; N = 3–5 per group) or for viral RNA measurements (N = 3–5 per group). Levels of viral RNA copies/ml were analyzed with the automated COBAS Ampliprep system V2 (Roche Molecular Diagnostics, Mannheim, Germany) as described^{31 (link)}. Brains were used for RNA extraction (N = 3 per group), immune-histochemical analyses (N = 2–3 per group) or isolation of brain infiltrating leukocytes (BILs; N = 5 per group).

Humanized mice were infected at 20 weeks of age with HIV NLEG1-70 GFP+ virus via intraperitoneal (i.p.) injection at a single dose of 10⁵ TCID50/ml. At 4 weeks and 10 weeks post-infection (w.p.i.), levels of viral RNA copies/ml were analyzed with the automated COBAS Ampliprep system V2 (Roche Molecular Diagnostics, Mannheim, Germany) as described19 (link).