Cell Culture, HIV-1
CD4+ T-cells were isolated from healthy human donor buffy coats by ficoll-paque density gradient followed by negative selection using a CD4+ isolation kit (Stem Cell Technologies, Vancouver Canada). Isolated CD4+ cells were cultured in RPMI medium containing 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin, referred to hereafter as complete RPMI. For stimulation, complete RPMI was supplemented with phytohemagglutinin (PHA-L) and interleukin-2 (IL-2). Following 3 days of stimulation, the media was changed to complete RPMI supplemented with only IL-2 to proliferate CD4+ T-cells.
HIV-1 BaL was obtained from the NIH AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, NIH. The HIV-1 strain BaL was used for infection of CD4+ T-cells consistent with guidelines from the NIH AIDS Reagent Program, and previous studies (33, 34).