Anti cd69 microbead kit 2

QVOA was performed as previously described [5 ,6 ]. Briefly, frozen PBMC were thawed and CD4+ cells were isolated using negative selecting bead purification (CD4+ T-cell Isolation Kit II, Miltenyi). The number of viable CD4+ T cells were counted and if the sample contained sufficient numbers rCD4 cells were isolated using negative selecting bead purification (anti-CD25, anti-biotin MicroBeads, anti-CD69 MicroBead Kit II, and anti–HLA-DR MicroBeads; Miltenyi). The resulting rCD4 cells were stimulated with Phytohaemagglutinin (PHA) and γ-irradiated PBMC and co-cultured with MOLT-4 cells transfected with CCR5 and naturally expressing CD4 and CXCR4 (MOLT4/CCR5; National Institutes of Health AIDS reagent program). The co-culture supernatants were tested for presence of HIV p24 by ELISA (PerkinElmer) after 14 or 21 days, to indicate outgrowth of replication-competent provirus [20 ]. For 2015 samples, all rCD4 cells were plated in a limiting dilution as previously described [20 ]. A portion of 2015 QVOA were tested for outgrowth viruses at 14 days, which was included and considered in both of the models. All time points collected after 2015, were measured at 21 days, with some also being tested at 14 days. In addition, QVOA for post-2015 samples were plated with a standard limited diluting plating strategy of approximately 14.5×10⁶ total rCD4 cells.