Dynabeads cd4 positive selection kit

Venous peripheral blood was obtained from healthy donors with known HLA types. Each blood donor had signed a written consent form, allowing for the use of his or her blood for research purposes. The procedure was approved by local ethics legislation. The blood was collected in heparinized tubes (Vacuette, Lithium Heparin, Greiner Bio One, Frickenhausen, Germany). Isolation of peripheral blood mononuclear cells (PBMCs) was accomplished by using Lymphoprep ™ gradient centrifugation (Axis-Shield, Oslo, Norway). The PBMCs were either used directly after the isolation or stored at −140 °C in a storage medium (RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA, USA), 40% heat-inactivated fetal calf serum (FCS) (Gibco), 10% dimethyl sulfoxide (Merck Millipore, Darmstadt, Germany), 100 U/mL penicillin/10 µg/mL streptomycin (Ampliqon, Odense, Denmark)). The CD4+ T cells were isolated from PBMCs using either a Dynabeads^® CD4 Positive Selection kit (Invitrogen, Life Technologies, Carlsbad, CA, USA) or a Dynabeads^® Regulatory CD4+ CD25+ T Cell Kit (Invitrogen), using only the CD4+ isolation part. The entire procedure was carried out according to the manufacturer’s guidelines. The purity of the isolated cells was evaluated by flow cytometry.

Uninfected bystander cells (GFP⁻ CD4^high T cells) were removed from the target cell population using the Dynabeads CD4-positive selection kit (Invitrogen) at a ratio of 25 μl of beads per million cells. Enrichment of infected primary GFP⁺ CD4^low T cells was assessed by cell surface staining with the anti-CD4 OKT4 Ab (Fig. 5A). Uninfected bystander cells were then replaced by the same number of autologous mock cells prior to staining with A32 or performing ADCC measurements.