Xvivo 15 without phenol red

Immediately after isolation, CD14⁺ or CD1a⁺ selected cells were treated with 17β-estradiol (E₂; Sigma) for 24h and washed before HIV exposure. E₂ was dissolved in 100% ethanol at an initial concentration of 1×10^-3M prior to evaporation to dryness and resuspension in Xvivo 15 without Phenol Red (Invitrogen) media containing 1% of charcoal dextran-stripped human AB serum (Valley Biomedical) to a E₂ concentration of 1×10^-5M. Dilutions were made to achieve a final working hormone concentration of 5×10^-8M. As a control, an equivalent amount of 100% ethanol without dissolved hormone was initially evaporated. All control conditions contained evaporated ethanol as a control. Because dextran charcoal treatment of serum may introduce LPS contamination, expression of costimulatory molecules after incubation of cells with or without 1% charcoal-stripped human AB serum was assessed (supplementary figure 2c).

Following ficoll purification, DCs were isolated using positive magnetic bead selection with either the CD14⁺ or CD1a⁺ isolation kits (Miltenyi Biotec) according to the manufacturer's instructions. After two rounds of positive selection, purity of the CD14⁺ and CD1a⁺ population was about 90% (see Supplementary Figure 3e). Isolated DCs were plated (20,000-100,000 cells/well) in round bottom ultra-low attachment 96-well plates (Corning, Corning, NY) in Xvivo15 without Phenol Red (Invitrogen) supplemented with 1% charcoal dextran-stripped human AB serum (Valley Biomedical) for in-vitro stimulation with HIV and hormones (E₂). No changes in phenotypic markers were observed after bead selection, except for a small decrease in CD14 MFI (Supplementary Figure 3c-d). The rate of cell recovery per gram of tissue after magnetic selection is shown in supplementary Figure 3a-b.
Cell morphology was evaluated after cytospin and Giemsa staining of CD1a and CD14 selected cells. Images were acquired using a IX73 Inverted Microscope (Olympus) with a 40× objective.