Quantifying HIV DNA in CD4 T cells

For measurement of HIV DNA in bulk CD4 T cells, CD4 T cells were enriched from cryopreserved PBMCs as above or using Dynabeads Untouched Human CD4 T Cell Enrichment kit (Invitrogen). DNA was extracted from PBMCs or enriched CD4 T cells using QIAamp Blood Mini Kit (Qiagen) and sorted CD4 T cell subsets (QIAmp DNA Micro Kit or Mini Kit) for use as input for qPCR assays. Copies of HIV-1 DNA were quantified and normalized to number of input cells (as determined by albumin PCR), by a previously described assay (41 (link), 42 (link)) with both qPCR assays performed in triplicate. For sorted populations PCR reactions for both albumin and total HIV were performed in triplicate for the CD32- population and in duplicate for the remaining populations, except where otherwise noted. Negative sample wells were replaced with zeros when averaging replicate values.