All target cells were stained with the green fluorescent cytosolic cell dye carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific, St. Laurent, QC, Canada) to distinguish them from PBMCs or NK effector cells. CFSE staining was performed per the manufacturer’s instructions and as previously described (55 (link)).
For binding experiments and ADCC assays using two target cells combined (i.e., rgp120-coated CEM cells or infected CEM cells 4 days postinfection (p.i.) with HIV combined with uncoated or uninfected CEM cells), CFSE+ cells were also stained with PKH26 red fluorescent membrane cell dye (PKH26 red fluorescent cell linker kit; Sigma-Aldrich) to distinguish them from CFSE+ PKH26− uncoated or uninfected CEM control cells, used as an internal control for nonspecific binding and killing. PKH26 staining was performed as previously described (55 (link)).
For binding experiments and ADCC assays using two target cells combined (i.e., rgp120-coated CEM cells or infected CEM cells 4 days postinfection (p.i.) with HIV combined with uncoated or uninfected CEM cells), CFSE+ cells were also stained with PKH26 red fluorescent membrane cell dye (PKH26 red fluorescent cell linker kit; Sigma-Aldrich) to distinguish them from CFSE+ PKH26− uncoated or uninfected CEM control cells, used as an internal control for nonspecific binding and killing. PKH26 staining was performed as previously described (55 (link)).
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