Secretory Leukocyte Protease Inhibitor Binds to Annexin II, a Cofactor for Macrophage HIV-1 Infection

Ma, Ge; Greenwell-Wild, Teresa; Lei, Kejian; Jin, Wenwen; Swisher, Jennifer; Hardegen, Neil; Wild, Carl T.; Wahl, Sharon M.

Flow Cytometry. Cells were incubated with 10 g/ml rhSLPI for 60 min in DMEM at 37 Њ C, washed, and stained using 1 g/ ml of goat polyclonal anti-SLPI (R&D Systems) for 30 min at 4 Њ C. Antibody-treated cells were rinsed, incubated with FITCconjugated secondary antibody (donkey anti-goat; Jackson Im-munoResearch Laboratories), washed, and fixed in 2% paraformaldehyde (Electron Microscopy Sciences) before analysis on a FACSCalibur flow cytometer (Becton Dickinson) using CELL-Quest software (21, 22) . Additionally, cells were incubated with 1 g/ml of biotinylated rhSLPI for indicated intervals, washed, and then treated with streptavidin-FITC (Pierce Chemical Co.) in staining buffer (PBS containing 0.5% BSA and 0.01% sodium azide). For additional staining, elutriated T cells, monocytes, and monocyte-derived macrophages detached from plastic with cell dissociation buffer (Invitrogen) were suspended in PBS with 2% FBS and stained with 10 g/ml of polyclonal goat annexin II (Santa Cruz Biotechnology, Inc.) and a secondary antibody (donkey anti-goat FITC; Jackson ImmunoResearch Laboratories). Single and double labeling with PE-CD14, FITC-CCR5, PE-CD11c, or PE-CD4 (BD Biosciences) confirmed cell specificity in flow cytometry analysis.