Rosettesep cd4 enrichment cocktail

CD4⁺ T cells were isolated from 3 to 4 ml of fresh ACD-anticoagulated whole blood using the RosetteSep CD4 Enrichment Cocktail (StemCell Technologies, Vancouver BC, Canada), following the manufacturer's instructions. Briefly, 50 μl of RosetteSep reagent was mixed per milliliter of whole blood, incubated at room temperature (RT) for 20 min, and then layered on Ficoll-Hypaque for density centrifugation. The layer of CD4⁺-enriched cells was washed twice with PBS and resuspended in 2 ml of RPMI containing 20% FCS and 20 IU/ml of IL-2 (Roche, Basel, Switzerland; 10799068001). Cells were counted using TruCount tubes (BD Biosciences, San Jose, California, USA) and simultaneously analyzed for purity, by flow analysis on a 5-laser Fortessa as previously described [42 (link)]. Typically, 1.5–2 × 106 cells at more than 96% purity of CD45⁺CD4⁺ cells were obtained and incubated in two wells of a 48-well plate (Corning, Corning, New York, USA). One well was left with IL-2 only, and to the second well was added 5 μl of anti-CD3/CD28/CD2 (StemCell Technologies). Cells were cultured for 3 days in 5% CO₂ at 37 °C, before RNA extraction, using the Maxwell RSC SimplyRNA Tissue kit. HIV-1 RNA transcript numbers in these cultures were normalized to copy numbers/input 10⁶ CD4⁺ T cells.