Hi fbs serum hi fbs

Fetal-derived Normal Human Astrocytes (NHAs, Lonza, Walkersville, MD) were maintained in Astrocyte Growth Media (AGM) BulletKit (Lonza). Early passages (1–4) were used in these experiments. U138MG astrocytoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA), and propagated in Dulbecco's modified eagle's medium (DMEM; Thermo Fisher, Waltham, MA) supplemented with 10% HI-FBS serum (HI-FBS; Sigma, St. Louis, MO) and 1% penicillin-streptomycin (Thermo Fisher). Cells were maintained in a 5% CO₂ humidified atmosphere at 37°C. Astrocytes were either primed with IFNγ (100ng/ml, R&D Systems, Minneapolis, MN) overnight then infected with HIV or HIV_VSV at 10ng/ml of p24 as indicated. Twenty-four hours post infection cells were washed and treated with new media. Seven days post infection; adherent cells were washed and removed by treatment with 0.24% (v/v) trypsin for 5 min, washed and suspended in sterile PBS for injection.

PBMCs were isolated from HIV sero-negative human donor venous blood and isolated using SepMate PBMC Isolation (STEMCELL Technologies). Cells were cultured overnight with 20u/ml IL-2 (NIH AIDS Reagent Program) and 1 μg/mL soluble α-CD3 and α-CD28 antibodies (BD Biosciences) in Roswell Park Memorial Institute medium 1640 (RPMI; Thermo Fisher) supplemented with 10% HI-FBS serum (HI-FBS; Sigma) and 1% penicillin-streptomycin (Thermo Fisher). Cells were maintained in a 5% CO₂ humidified atmosphere at 37°C. PBMCs were then infected with 2ng/1×10⁶ cells/ml of HIV-1-GFP. Cells were washed and maintained for one week in culture before reconstitution of 2 × 10⁷ cells per neonatal animal xenotransplanted with uninfected NHAs.