X vivo 15 chemically defined medium

To examine the inhibition effect of human SP on HIV-1 infection, 1 × 10⁵ TZM-bl cells/ml were seeded into 96-well flat-bottom plates (100 μl). The next day, the medium of the cells was replaced by X-VIVO 15 chemically defined medium (Lonza) supplemented with 0.05 mM aminoguanidine (41 (link)) and gentamicin (50 μg/ml). SP diluted in PBS was added to cells and incubated for 1 hour at 37°C. After incubation, cells were infected with X4-tropic or R5-tropic HIV-1 by spinoculation (30 min, 2090g, 37°C) and incubated for another 2 hours. Final inoculum was discarded, cells were washed with PBS, and 200 μl of X-VIVO 15 medium [+ 0.05 mM aminoguanidine, gentamicin (50 μg/ml)] was added. Infection rates were measured 2 days after infection using a Gal-Screen system (Applied Biosystems).

To examine the influence of polyamines on X4-tropic and R5-tropic HIV-1 infection, 1 × 10⁵ TZM-bl cells/ml were seeded into 96-well flat-bottom plates (100 μl). The next day, the medium was changed to 170 μl of X-VIVO 15 chemically defined medium (Lonza) and 20 μl of substances was added. TZM-bl cells were incubated for 1 hour at 37°C before they were inoculated with 10 μl of virus (cell treatment). Alternatively, the virus was treated for 15 min with the polyamines before 20 μl of the mixture was added to TZM-bl cells in 180 μl of X-VIVO 15 medium, thereby diluting the mixture 10-fold. To analyze SP-derived library fractions, TZM-bl cells in 85 μl of X-VIVO 15 medium were preincubated with 5 μl of the respective fraction. Cells were inoculated with 10 μl of virus after 30 min. Infection rates were measured 2 days after infection using a Gal-Screen system (Applied Biosystems).

PAP248-286 was purchased from Celtek Peptides. SEVI was prepared by dissolving the peptide at 2.5 mg/ml in PBS and shaking at 2000 rpm on a rotary shaker for 2 days at 37°C. For infection experiments, 1 × 10⁵ TZM-bl cells/ml were seeded into 96-well flat-bottom plates (100 μl). The next day, the medium was changed to 170 μl of X-VIVO 15 chemically defined medium (Lonza) and 20 μl of substances was added for 1 hour. The virus was 1:1 mixed with SEVI or PBS and preincubated for 10 min at room temperature before cells were inoculated with 10 μl of the mixture. Infection rates were measured 2 days after infection using a Gal-Screen system (Applied Biosystems).