Nkg2a pe

Peripheral blood monouclear cells (PBMCs) were isolated from patients and healthy individuals, suspended in RPMI 1640 medium containing 2 mM/L L-glutamine and 10% fetal calf serum (Hyclone). Isolated PBMCs were stained with fluorochrome-conjugated antibodies to CD3-PerCP/cy5.5, CD56-FITC, CD8-FITC and CD4-FITC (Biolegend, San Diego, CA). NK cells surface receptors were stained with fluorochrome-conjugated antibodies to NKp46-PE, NKG2A-PE, NKp30-APC, NKG2C-APC, NKG2D-APC and isotype matched controls (Biolegend, San Diego, CA). Stained PBMC were isolated using a FACS Calibur flow cytometer (Becton Dickinson, USA) and analyzed by FlowJo analysis software (Treestar, Ashland, OR).

In order to analyze NK cell subsets and their receptor expression profile, PBMCs were labeled with anti-CD3-BV785, -CD16-BV570, -CD38-BV510, -CD56-BV711, -CD57-FITC, -NKG2A-PE, -NKG2D-PE/Cy7, -NKp30-BV421, -NKp44-PE/Cy7 and -NKp46-AlexaFluor700 mAbs (all from Biolegend, San Jose, CA, USA) for 20 minutes at room temperature in the dark. The data were collected and analyzed with NovoCyte flow cytometer with NovoExpress operating system software (Agilent Technologies, USA).