Figure S1. Assay for virion production.... | Rockefeller University Press

Figure S1.

$Assay for virion production. (A) Detection of HIV-1 virions in culture supernatants. To determine the sensitivity of the assay for supernatant virions, HIV-1 pseudoviruses were generated by transfection, pelleted by ultracentrifugation, and quantified using a p24 ELISA. The indicated number of pseudoviruses were subjected to one freeze/thaw cycle, added to a volume of culture media equal to that of the pooled supernatants harvested from a single culture well, and processed by the same steps of ultracentrifugation, RNA isolation, and quantification used for the culture supernatants (see Materials and methods). Graph shows the fraction of replicates with detectable HIV-1 RNA as a function of the input number of pseudoviruses. 16 experimental replicates were analyzed per condition. (B) Use of an internal standard to monitor recovery of viral RNA. To verify that wells negative for HIV-1 RNA had low or absent virion production, we measured the RCAS internal standard virus in each well (Palmer et al., 2003). BLD = below limit of detection. (C) Histogram displaying the distribution of RCAS internal standard virus qPCR cycle thresholds (Ct) after viral RNA extraction. Samples were considered to have normal recovery (blue) or poor recovery (red) based on visual inspection of the curve. Samples with poor RNA recovery were excluded from further analysis.$

Assay for virion production. (A) Detection of HIV-1 virions in culture supernatants. To determine the sensitivity of the assay for supernatant virions, HIV-1 pseudoviruses were generated by transfection, pelleted by ultracentrifugation, and quantified using a p24 ELISA. The indicated number of pseudoviruses were subjected to one freeze/thaw cycle, added to a volume of culture media equal to that of the pooled supernatants harvested from a single culture well, and processed by the same steps of ultracentrifugation, RNA isolation, and quantification used for the culture supernatants (see Materials and methods). Graph shows the fraction of replicates with detectable HIV-1 RNA as a function of the input number of pseudoviruses. 16 experimental replicates were analyzed per condition. (B) Use of an internal standard to monitor recovery of viral RNA. To verify that wells negative for HIV-1 RNA had low or absent virion production, we measured the RCAS internal standard virus in each well (Palmer et al., 2003). BLD = below limit of detection. (C) Histogram displaying the distribution of RCAS internal standard virus qPCR cycle thresholds (Ct) after viral RNA extraction. Samples were considered to have normal recovery (blue) or poor recovery (red) based on visual inspection of the curve. Samples with poor RNA recovery were excluded from further analysis.