Round bottom ultra low attachment 96 well culture plates

Following removal of dead cells, CD4+ T cells were isolated by negative magnetic bead selection with the CD4+ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications, as previously described^{35 (link)}. Anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension. After two rounds of negative selection, purity of the CD4+ T cell population was higher than 90%. Isolated CD4+ T cells were activated for 24 hr with PHA and IL-2 as described for blood CD4+ T cells. Activated CD4+ T cells were plated at a density of 1 × 10⁵ cells per well in round-bottom ultra-low attachment 96-well culture plates (Corning, Corning, NY) in 0.2 ml of Immune Cell Media.

Blood Leuko Paks from women were obtained from our IRB-approved collection facility at Dartmouth-Hitchcock Medical Center. CD4+ T cells were purified with the CD4+ T cell isolation kit (Miltenyi Biotech) following isolation of peripheral blood mononuclear cells (PBMC) by standard Ficoll density gradient centrifugation^{35 (link),56 (link)}. Freshly isolated blood CD4+ T cells were activated in vitro using Xvivo 15 media with Phenol Red plus Phytohemagglutinin (PHA, 2.5 µg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml, AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann- La Roche Inc.) for 24hr as described previously^{56 (link)}. Activated CD4+ T cells were plated at a density of 1 × 10⁵ cells per well in round bottom ultra-low attachment 96-well culture plates (Corning, Corning, NY) in 0.2 ml of Immune Cell Media consisting of X-VIVO 15 Media (Lonza, Walkersville, MD) supplemented with 10% charcoal stripped human AB serum (Valley Biomedical, Winchester, VA) prior to treatment.