Dynabeads untouched human cd4 t cells kit

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Corning) with 10% fetal bovine serum (FBS) (Sigma-Aldrich), Jurkat cells and peripheral blood mononuclear cells (PBMCs) were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Corning) with 10% FBS at 37°C and 5% CO₂. Resting CD4+ T cells were purified from bulk PBMCs by using Dynabeads Untouched Human CD4 T Cells Kit (Thermo Fisher Scientific). Selected CD4+ T cells were activated by Dynabeads Human T-Activator CD3/CD28 kit (Thermo Fisher Scientific) and were maintained in RPMI 1640 with 10% FBS, containing 30 U/ml IL-2 (Sigma-Aldrich).

To determine whether sequence-specific differences in RNA stability could contribute to differences in levels of the HIV RNAs, we measured the decay of Read-through, TAR, Long LTR, Nef, PolyA, and MS Tat-Rev transcripts in CD4+ T cells from an ART-suppressed individual using RNA Pol II inhibitors, Triptolide and Actinomycin D. CD4+ T cells were isolated from blood from an ART-suppressed individual using the Dynabeads Untouched Human CD4 T cells kit (Thermo Fisher, Waltham, MA). Replicate aliquots of CD4+ T cells (6x10⁶ cells/well) were seeded into 6-well tissue culture plates (Corning Inc., Corning, NY) at a concentration of 1x10⁶ cells/ml in complete RPMI with either DMSO (negative control), 100nM Triptolide (Sigma, St Louis, MO), or 5mg/ml Actinomycin D (Sigma, St Louis, MO). Cells were harvested at the following time points: DMSO: 0, 1 and 16h; Triptolide: 0, 1, 3, 6 and 16h; Actinomycin D: 0, 1, 3 and 16h. HIV transcripts (Read-through, TAR, Long LTR, Nef, Poly A, and MS Tat-Rev) were quantified using RT-ddPCR as described above. Levels of each HIV RNA were quantified by RT-ddPCR, normalized by alternative measures (cell counts, DNA mass, RNA mass), and expressed as a fraction of the value at time zero. The half-life for each transcript was determined using an exponential one-phase decay model.