Anti cd107a

Antibody-dependent NK cell degranulation and cytokine/chemokine secretion was measured using freshly isolated NK cells, after the culture of rhesus macaque plasma with plate-bound Env gp120. Proteinbinding plates were coated with Env gp120 (300 ng/well) and incubated for 2 hr at room temperature. Plates were then blocked in 5% BSA overnight at 4°C. Fresh NK cells were isolated from whole blood from seronegative donors using negative selection with RosetteSep, as recommended by the manufacturer. Plates were washed to remove unbound antigen, sample plasma was added and incubated for 2hr at 37°C. Following the incubation, plates were washed to removed un-opsonized antibodies. Isolated primary NK cells with anti-CD107a, brefeldin A (10 mg/ml) (Sigma), and GolgiStop (BD) were added, and incubated for 5 hours at 37°C. The cells were then washed and stained for surface markers using anti-CD16, anti-CD56, and anti-CD3. The cells were then washed, fixed and permeabilized using Fix & Perm (Invitrogen), and then stained 29 intracellularly with anti-IFN-γ and anti-MIP-1β. The cells were then fixed in 4% paraformaldehyde and analyzed using flow cytometry. NK cells were defined as CD3- negative and CD16- and/or CD56-positive. All antibodies for flow cytometry were purchased from BD.