Activation of Primary CD4+ T Cells

Primary human cells from HIV seronegative individuals were cultured in “RF10 medium” consisting of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Omega Scientific), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen). PBMCs were isolated using Ficoll-Paque Plus separation (GE Healthcare). Primary CD4+ T cells were separated from PBMCs by negative immunomagnetic selection using the CD4+ T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were then exposed to compound for 24 hours before staining and flow cytometric analysis of CD69 levels. Compound incubations were performed by seeding 10⁵ cells/well in 100 μL of RF10 media containing the appropriate concentration of compound in wells of a v-bottomed 96-well plate. Bryostatin 1 (Tocris bioscience) and Prostratin (LC Laboratories) were obtained commercially, and bryologs or Prostratin analogs were synthesized as previously described [18 (link),26 (link)].