Ccr5 fitc

Single-cell suspensions from blood, omentum, and liver were stained with CD3-APC-Cy7 (BioLegend, San Diego, CA, USA) and CD56-APC, CD56-FITC, NKP46-PE, NKP30-APC, NKG2D-APC, CCR5-FITC, CXCR3-PE, CCR1-APC, CCR2-APC, CXCR2-FITC, CCR3-PE, and CCR6-APC (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were quantified as CD56 + CD3 2 cells within the lymphocyte gate. For cytokine profiling and degranulation assay, cells were stimulated with PMA (10 ng/ml) and ionomycin (1 mg/ml) for 1 h. PE-conjugated anti-CD107a (BD Biosciences UK, Oxford, United Kingdom) was added at this time to detect degranulation, followed by the addition of brefeldin A (10 mg/ml) for a further 3 h. Subsequent staining with IFNg-V500, TNF-a-APC, and IL-10-PE (BD Biosciences UK) was performed. Cells were acquired using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).