17β-estradiol (E2, Calbiochem, Gibbstown, NJ) and progesterone (P, Calbiochem) were dissolved in 100% ethanol for an initial concentration of 1 × 10− 3 M, evaporated to dryness and suspended in X-VIVO 15 without Phenol Red media supplemented with 10% charcoal dextran-stripped human AB serum (Valley Biomedical) to a concentration of 1 × 10− 5 M. Further dilutions were made to achieve final working concentration, and cells were treated with 5 × 10− 8 M E2 or 1 × 10− 7 M P. Both are standard hormone treatment concentrations used by our laboratory and each is within the physiological range of hormone concentrations in the FRT [52 (link)]. As a control, an equivalent amount of ethanol without dissolved hormone was initially evaporated.
Charcoal dextran stripped human ab serum
Manufactured by Valley Biomedical
Charcoal dextran-stripped human AB serum is a laboratory product used to remove steroid hormones and other lipophilic compounds from biological samples. It is a cell culture supplement derived from human AB blood serum that has been processed to remove these compounds.
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5 protocols using charcoal dextran stripped human ab serum
1
Hormone Treatment Protocol for FRT
2
Estrogen Priming Enhances HIV Susceptibility
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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 ).
3
Estrogen Priming Enhances HIV Susceptibility
4
Isolation and Characterization of Dendritic Cells
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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.
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.
5
Isolation and Characterization of Dendritic Cells
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