Nkg2a z199

Mononuclear cells from spleen of SIV-naïve RM were stained with primary antibody staining which included: CD3 (clone SP34.2; manufacturer BD Pharmingen), CD14 (M5E2; BD Pharmingen), CD20 (2H7; BioLegend), HLA-DR (G46-6; BD Pharmingen), and NKG2A (Z199; Beckman Coulter). After incubation for 20 minutes at room temperature, the cells were washed with wash buffer (1XPBS containing 2% FBS) and stained with DAPI dye (ThermoScientific) for live and dead cell discrimination. After 5 minutes of incubation, cells were washed and fixed with 1% formaldehyde. Samples were recorded using an ImageStreamX Mk II (EMD Millipore) and analyzed using IDEAS Application v6. The general analysis, colocalization and internalization modules in the IDEAS software were utilized in analyzing these samples.

To immobilize different antibodies in a comparable way to E-plates, we decided to coat the wells first with goat-anti-mouse antibodies. Therefore, E-Plates 16 PET were pre-coated with 50 μl of 5 μg/ml goat-anti-mouse antibody for 3 hours at 37 °C and subsequently washed three times with PBS. In pilot experiments we determined that the subsequent coating with an antibody against NK cell surface receptors gave the strongest signal (Fig. S1). In contrast, binding the antibody first to the NK cells or adding the antibody at the same time as the NK cells without washing was less effective (Fig. S1). Therefore, primary antibody was added to the pre-coated wells at 2 μg/ml in a volume of 50 μl and incubated at 37 °C for 1 hour. Plates were washed three times with PBS and background reading was performed in 100 μl medium.
The following antibodies were used: CD16 (3G8), 2B4 (C1.7), NKp46 (9E2), NKp80 (5D12), CD28 (28.2), DNAM-1 (TX25), CD56 (NCAM) (all from Biolegend), CD3 (OKT3, eBioscience), NKG2D (R&D), NKp30 (p30-15, produced in our laboratory), NKp44 (p44-2, produced in our laboratory), Mouse IgG1 (MOPC-21, Sigma), CD45 (BD Transduction laboratories), NKG2A (Z199, Beckman Coulter), goat anti-mouse (dianova), and CD94 (HP-3D9, BD Bioscience).