Cd4 tx red

Cryopreserved lymphocytes isolated from the blood samples obtained from HIV-infected or normal patients were quickly thawed in a 37°C water bath and washed with PBS. Cells were then stained for cell surface markers using specific antibodies. The immune activation panel consisted of antibodies CD3-Cy7, CD4-Tx red, CD8-APC along with immune activation markers CD38 PE and HLA-DR FITC (BD Pharmingen) (Figure S1A). The apoptosis panel comprised of the following antibodies CD3-Cy7, CD4-Tx red, CD8-APC (Beckman Coulter) along with CaspACE FITC-VAD-FMK (Promega) (Figure S1B). Stained cells were washed and fixed using Cytofix reagent (Beckman coulter) and acquired on a 10 color Beckman Coulter Gallios flow cytometer. At least 20,000 events for each sample were acquired. Data was analyzed using FlowJo software (Tree Star). Cells were first gated on CD3+ population and immune activation/apoptosis on CD4+ and CD8+ T cell subsets determined (Figure S1A & B). For in vitro T cell activation, lymphocytes were cultured in RPMI-1640 medium supplemented with 20% FBS and Phytoheamagglutinin (Sigma) at 2.5 μg/ml and IL-2 (Roche) at 10U/ml for 48h, stained and analyzed as above for immune activation markers and CD4:CD8 ratios (Figure S2).