Neutralization kinetics of cell-free vector.

<p>(<b>A</b>) Vector particles were incubated in escalating concentrations of anti-VSV-G neutralizing antibody for 1 hour at 4°C in the presence of 4 µg/ml protamine sulfate. Particles were then placed on 293T cells for overnight transduction at 37°C. Cells were washed after 24 hours, and FACS was performed 48 hours later. (<b>B</b>) Experiment was performed as described in (A) with the exception that mouse serum was used instead of VSV-G neutralizing antibody and the neutralizing incubation was performed at 37^°C for the purpose of minimizing cell death resulting from serum incubation (CD). In the cell-bound condition (black squares), 1x10⁵ prechilled Jurkat cells were exposed to vector (<b>C</b>: MOI 0.3; <b>D</b>: MOI 3) for 1 hour at 4°C in the presence of 4 µg/ml protamine sulfate, then placed in co-culture with 2.5x10⁴ pre-plated 293T cells at 37^°C. Cell-free samples (gray circles) were prepared in parallel, in the absence of Jurkat cells. Escalating concentrations of anti-VSV-G neutralizing antibody were added to co-cultures and incubataed overnight at 37°C. Cells were then washed, and FACS was performed 48 hours later (EF). 1x10⁵ prechilled Jurkat cells were exposed to vector (MOI 3) for 1 hour at 4°C, then placed in co-culture with 2.5x10⁴ pre-plated 293T cells at 37°C. At serial time points (<i>x</i>-axis), 0.5% anti-VSV-G neutralizing antibody (E) or 10% mouse serum (F) was added to the transduction culture. Target 293T cells were washed 24 hours after vector exposure, and FACS was performed 48 hours later. CD45-APC antibody was used to exclude primary Jurkat cells (gray circles) from secondary target 293T cells (black circles). Red line depicts % 293T GFP+ events/ % Jurkat GFP+ events.