Anti kir3dl1 apc dx9

DNA was extracted from whole blood using a Roche MagNAPure 96 or Qiagen® DNeasy Blood & Tissue kit according to the manufacturer’s instructions. KIR3DL1 typing was performed using PCR primers described by Boudreau et al. to detect the major alleles *004 (null), *001, *002 (high), *013 (3DS1) and *005, *007 (low). [25 (link)]. Furthermore, flow cytometry was used to determine the KIR3DL1 expression level by staining cells with anti-CD3-APC-H7 (SK7), anti-CD14-APC-H7 (MϕP9), anti-CD16-BV786 (3G8), anti-CD56-BV711 (NCAM1), anti-CD57-BV605 (NK-1), anti-CD19-APC-H7 (SJ25C1; all from BD Biosciences), anti-NKG2C-Alexa Fluor 488 (134,591), anti-KIR2DL1 Alexa Fluor 700 (143211, both R&D Systems), anti-NKG2A-PE (Z199), anti-KIR2DL1/S1-Pe-Cy7 (EB6B), anti-KIR2DL2/L3/S2-Pe-Cy5.5 (GL 183; all from Beckman Coulter), anti-KIR3DL1-APC (DX9; BioLegend). Stained samples were analyzed on a five laser BD LSR Fortessa SORP instrument. Data was analyzed using FACSDiva (v.8.0.1 or later) and FlowJo (v.10.8.1 or later) software (BD Biosciences). The median frequency of KIR3DL1⁺ cells among CD16⁺CD56⁺ NK cells was 11% (range 7.71–56.5%). The HLA-B and -C allele genotype was determined using LABType SSO Class I Locus Typing Tests from One Lambda as described elsewhere [26 (link)]. Complementary KIR ligand typing for HLA-A Bw4 was performed using the Olerup SSP KIR HLA Ligand kit.

NK cells were cultured overnight with 100 IU/ml of IL-2 (Proleukin, Novartis Pharmaceuticals) or non-stimulated (resting) in 0.1 million cells/well before addition of 0.1 million K562 cells/well. Anti-CD107a-BUV395 (H4A3, BD Biosciences) was added to measure the degranulation. After 4 h-incubation, cells were stained with anti-CD56-BV711 (NCAM 16.2, BD Biosciences), anti-NKG2A-PE (Z199), anti-KIR2DL1/S1-PE-Cy7 (EB6B), anti-KIR2DL2/L3/S2-PE-Cy5.5 (GL183; all Beckman Coulter) and anti-KIR3DL1-APC (DX9, Biolegend). Gating strategy is shown in Supplementary Fig. 1.
In intracellular cytokine assays, 50 000 NK cells/well were cultured and stimulated overnight, using same conditions as in degranulation assays. A five-hour co-culture with 50 000 K562 cells/well at 37 °C was performed, adding Brefeldin A (GolgiPlug, BD Biosciences) after one hour. The cells were stained with the same antibody panel as mentioned above, followed by fixation and permeabilization with BD Cytofix/Cytoperm (BD Biosciences). At the end, cells were stained with anti-IFNγ-BUV395 (B27) and anti-TNFα-AF488 (Mab 11; both BD Biosciences).
Stained samples were analyzed using a five laser BD LSR Fortessa SORP instrument. Data was analyzed using FACSDiva (v.8.0.1 or later) and FlowJo (v.10.8.1) software (BD Biosciences).