Surface staining of rVSV-EnvG<sub>4</sub>-G<sub>6</sub>-infected Vero cells and rVSV-EnvG<sub>4</sub>-G<sub>6</sub> particles.

<p>(a) After a 22 hr infection, Vero cells were detached from plate by gentle trypsin treatment and resuspended in PBS. 5×10<sup>6</sup> cells were analyzed for VSV G and HIV-1 EnvG surface expression. All cells analyzed for Env staining were first gated as positive for G staining. (b) For rVSV staining, 10<sup>9</sup> pfu of virus was bound to alum at 37°C with agitation. rVSV/alum conjugates were stained with titrated anti-VSV-G (Vi10) or anti-HIV-1 Env Ab followed by anti-human IgG or anti-mouse IgG2a Alexa555 and acquired on a modified LSRII flow cytometer. Median fluorescent intensity (MFI) was determined for each Ab dilution. (c) 10<sup>9</sup> pfu of virus was incubated with SYTO 63 nucleotide stain in PBS for 30 min at RT followed by incubation with anti-VSV-G (Vi10) and then anti-mouse IgG2a Alexa555. Virus was analyzed as described above. Minimum threshold settings on SSC were used to increase sensitivity for small particles and FSC and SSC parameters were set to log scale. Deionized water was run for 15 min to equilibrate for low threshold noise. ∼50,000 events were acquired for the PBS control, ∼10<sup>6</sup> events were acquired for virus samples. Particles staining positive for nucleic acid that were above the noise threshold were gated on, and the amount of anti-G staining for those populations were compared.</p>