Figure 2.
Binding of VSV-G- or scFv/VSV pseudotyped HIV-1 particles to target cells. 5 × 105 293T or Balb/C cells were incubated with 1 ml (non-concentrated) pseudotyped HIV-1 particles from transiently transfected 293T cells for 30 minutes on ice. Cells were washed twice with phosphate buffered saline (PBS, pH 7.0) and incubated in block buffer (BB: 10% bovines serum albumine, 0.1 M Glycine in PBS (pH 7.0)) for 30 minutes on ice, which was then replaced by 200 μl of 5G8F11 hybridoma supernatant, kindly donated by Dr Douglas Lyles (Winston-Salem NC, US). After 1 hour on ice, the cells were washed twice with BB and resuspended in 100 μl fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (FITC-Ab) (Sigma), diluted 100 times in BB. The cells were rinsed again after 1 hour, fixed with 0.2 % formaldehyde and analysed using a FACScalibur fluorescence-activated cell sorter (Becton Dickinson).