Cd158a fitc

The following mAbs were used to detect surface markers: anti-CD3 APC-Cy7 (clone: UCHT1), CD14 APC-Cy7 (HCD14), CD16 BV510 (3G8), CD19 APC-Cy7 (HIB19), CD25 BV421 (BC96), CD69 BV421 (FN50), CD56 PE-Cy7 (HCD56), CD4 FITC (RPAT4) and CD62L (DREG-56), all IgG1 isotypes from Biolegend. CD57 VioBlue (TB03. isotype IgM) CD159C PE (REA 205, isotype IgG1), CD279 PE (PD1.3.1.3. isotype: IgG2B), CD8 Vioblue (REA734 isotype IgG1) and CD45RA PE-Vio770 (REA 562. isotype IgG1) were from Miltenyi Biotech Bergisch, Gladbach, Germany, CD158a FITC (HP-3E4 isotype IgM), CD158b FITC (CH-L. isotype IgG2b) and CD27 BV510 (L128. isotype IgG1) were from BD Biosciences. CA. USA. CD159a APC (Z199. isotype IgG2b) and CD336 PE (Z231. Isotype IgG1) were from Beckman Coulter, (Beckman Coulter. 250 S. Kraemer Blvd, Brea, CA 92821, USA).

The monoclonal antibodies CD3‐PerCP, CD56‐allophycocyanin(APC), CD158a‐FITC, CD158b‐PE, CD158e‐FITC, CD94‐FITC, CD62L‐FITC, CD54‐PE, CD11a‐FITC, CX3CR1‐FITC, CXCR4‐PE, CCR7‐PE, NKP30‐FITC, NKP46‐PE, IL13‐PE, IL10‐PE, TGF‐β‐PE and IFN‐γ‐FITC (BD Bioscience, Mountain View, CA, USA), and NKG2A‐PE (BeckmanCoulter, USA) and appropriate isotypes were used in individual 4‐colour flow cytometry assays to analyse the immunophenotype as well as the cytokine secretion of NK cells. Intracellular staining was performed using the Pharmingen Intracellular Staining Kit (BD Pharmingen, San Diego, CA, USA). The cells were incubated for 5 hours with phorbol myristate acetate (PMA) (40 ng/mL) plus ionomycin (2.5 μg/mL, all reagents from Sigma Chemical) to stimulate maximal IFN‐γ, IL‐13, TGF‐β and IL‐10 production; GolgiStop (0.7 μL/mL) was added to the sample during the last 4 hours to trap the protein in the cytoplasm. NK1, NK2, NK3 and NKr cells were identified as CD3⁻CD56⁺IFN‐γ⁺, CD3⁻CD56⁺IL‐13⁺,CD3⁻CD56⁺ TGF‐β⁺ and CD3⁻CD56⁺IL‐10⁺, respectively. The dose of NK1, NK2, NK3 and NKr cells was classified as the absolute number of NK1, NK2, NK3 and NKr cells infused in GBM and GPB (cells/kg). Data were analysed using a FACSCaliber 4‐colour flow cytometer (BD Biosciences) and FlowJo 7.6.1 software (Tree Star Inc, CA, USA).