Anti nkg2d apc mab clone 1d11

NKL cells (a commonly used immortal human NK cell line, derived from an aggressive human leukemia) were left uncoated or coated with 30, 60 or 120 μg/ml of rhMICA-Fc or rhULBP2-Fc (R&D Systems), labeled in-house with Atto488, for 30 min at 37°C. After incubation, cells were washed in PBS, 2% FBS, fixed in PBS, 4% PFA for 15 min, and stained with anti-NKG2D-APC mAb (clone 1D11, Biolegend), or isotype-matched control mAb (mouse IgG1 isotype control, clone MOPC-21, Biolegend, conjugated with APC) for 30 min at 4°C. Cells were then washed in PBS, 2% FBS, post-fixed in PBS, 2% PFA, and analyzed with a BD FACS Canto II flow cytometer (BD Biosciences). Data were analyzed with FlowJo_V10 software.

To assess the cell surface expression of NKG2D and IL-2/IL-15Rβ, cells were washed and blocked with phosphate-buffered saline (PBS), 2% fetal bovine serum (FBS) for 30 min at 4°C and then were stained with Zombie Aqua viability dye (Biolegend), anti-CD56-AF488 mAb (clone HCD56, Biolegend), and anti-NKG2D-APC mAb (clone 1D11, Biolegend) or anti-IL-2/IL-15Rβ-APC mAb (clone TU27, Biolegend), respectively, or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21, Biolegend, conjugated with AF488 or APC) for 30 min at 4°C. Cells were then washed in PBS, 2% FBS, fixed in PBS, 2% paraformaldehyde (PFA), and analyzed with a BD FACS Canto II flow cytometer (BD Biosciences). Data were analyzed with FlowJo_V10 software.