A cell-free virus stock of CCR5-tropic HIV-1 clone Bal was obtained from the AIDS Research and Reference Reagent Program (ARRRP), Division of AIDS, NIAID, NIH. HIV-1 Bal virus was propagated in human peripheral blood mononuclear cells and harvested 10 days' post-infection. Virus was titrated using the Alliance HIV-1 p24 ELISA kit (PerkinElmer, Waltham, MA).
Hu-NSG mice with stable human leukocyte reconstitution (with a 20% or higher percentage of human CD45+ T cells) were infected with the HIV-1 Bal (200 ng p24/mouse) by intraperitoneal injection (i.p.) while under inhalant general anesthesia. Plasma viremia was assayed using one-step reverse transcriptase real-time PCR 22 (link) with automated CFX96 TouchTM Real Time PCR Detection System (Bio-Rad). HIV-1 levels in peripheral blood were determined by extracting RNA from 5×105 cells using the QIAamp Viral RNa mini kit (Qiagen) and performing Taqman qPCR using a primer and probe set targeting the HIV-1 LTR region, using the TaqMan Fast Virus 1-Step Master Mix as per the manufacturer's recommendations. The limit of detection in this assay is 40 copies/mL or less, but due to dilution, the limit of detection of our assays was 500-800 copies/mL, depending on the available sample volume.
Hu-NSG mice with stable human leukocyte reconstitution (with a 20% or higher percentage of human CD45+ T cells) were infected with the HIV-1 Bal (200 ng p24/mouse) by intraperitoneal injection (i.p.) while under inhalant general anesthesia. Plasma viremia was assayed using one-step reverse transcriptase real-time PCR 22 (link) with automated CFX96 TouchTM Real Time PCR Detection System (Bio-Rad). HIV-1 levels in peripheral blood were determined by extracting RNA from 5×105 cells using the QIAamp Viral RNa mini kit (Qiagen) and performing Taqman qPCR using a primer and probe set targeting the HIV-1 LTR region, using the TaqMan Fast Virus 1-Step Master Mix as per the manufacturer's recommendations. The limit of detection in this assay is 40 copies/mL or less, but due to dilution, the limit of detection of our assays was 500-800 copies/mL, depending on the available sample volume.
