Anti cd158a

For analytical Flow Cytometry or preparative FACS sorting the following fluorescently labeled antibodies were used: anti-CD56 (clone B159), anti-CD16 (clone 3G8), anti-CD3 (clone SK7), anti-CD9 (clone ML-13), anti-CD49a (clone SR84), anti-CD151 (clone 14A2.H1), anti–granzyme A (clone CB9), anti-perforin (clone dG9), anti–granzyme B (clone GB11),and isotype controls were all from BD Biosciences (San Diego, CA). Cell surface molecule staining and intracytoplasmic staining were done as previously described[36 ]. KIR expression was evaluated with a cocktail of four PE-conjugated antibodies: anti-CD158b, anti-CD158a, anti-NKB1 and anti-NKAT2 all from BD Biosciences. Preparative FACS sorting was done using a FACS Aria cell sorter (Becton Dickinson). Analytical flow cytometry was performed on a BD FACSCanto flow cytometer (Becton Dickinson). Chemokine receptor staining was done as previously described[36 ] using the following fluorescently labeled mAbs or corresponding isotype controls: anti-CXCR4 (clone 12G5), anti-CXCR1 (clone 42705), anti-CXCR3 (clone 49801), anti-CCR7 (clone 150503) (all from R&D Systems), and anti-CX3CR1 (clone 2A9-1; MBL International, Woburn, MA). Data from CD56Dim idNK cells not previously shown is compared here to that of CD56Bright idNKs.

Lymphocytes from blood/tissue samples were incubated for 30 minutes with the following mouse anti-human monoclonal antibodies: anti-CD45 (Biolegend, CA, USA); anti-CD3, anti-CD56, anti-perforine, anti-CD158a, anti-CD158b and anti-NKp44; anti-NKG2D, anti-NKp30, anti-CD226, and anti-NKp46 (BD Bioscience, San Jose, CA); anti-NKG2A and anti-NKG2C (R&D Systems, Inc., Minneapolis, MN); KIR2DL1/DS1 and KIR3DL1/DS1 (Beckman Coullter, Fullerton, CA). Mouse serum was used to block non-specific Fc receptor (FcR) binding, and isotype-matched IgGs were used as negative control antibodies. The samples were acquired using an fluorescence activating cell sorter (FACS) Calibur flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).