Cd62l fitc

Flow cytometric analyses were conducted using an LSR II flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Version 7.6.5; Flowjo). All antibodies against human or mouse cells were used at appropriate dilutions, as determined by previous titration. Doublet discrimination was carried out and non-viable cells were excluded by 4,6 diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich). Mouse cells were characterized using the antibodies as follows: CD3-V500, CD4-V450, CD11c-APC Cy7, SiglecF-AF647, Ly6G-FITC, CD11b-V500, B7-1-V450, B7-2-PE Cy7, CD62L-FITC (BD Biosciences); mPDCA1-APC, B220-PE (Miltenyi Biotec); CD8-PE Cy7, F4/80-PerCP, SiglecH-PE, TLR4-AF488, TLR9-FITC, MHC-I-FITC, MHC-II-V450, PD-L1-PerCP, ICOS-L-PE, OX40L-APC (eBioscience); TLR7-PE (Abcam); p75NTR-AF488 (Advanced Targeting Systems), CD45-Pacific Blue (Biolegend), and panTrk-FITC (Cell Signaling Technology). Human cells were characterized using the antibodies as follows: TrkA-PE (R&D Systems); BDCA-2-FITC, BDCA-4-PE, p75NTR-APC, p75NTR-FITC, p75NTR-PE (Miltenyi Biotec); CD45-V500, CD3-PE, CD4-FITC, CD8-PerCP, FcεRIα-FITC, IL-3R-PE Cy7, CD184-PE Cy7, MHC-I-PE Cy7, MHC-II-PE, CD80-V450, CD86-PE, CD83-V500, OX40L-V500, PD-L1-PE Cy7, CCR7-V450, CCR9-APC (BD Biosciences), CD3-APC eFluor780, CD4-APC, CD8-PE Cy7, CD25-PE (eBioscience), and CD69-PerCP Cy5.5 (BioLegend).

Blood cells from 300 μl blood were incubated in RBC-Lysis buffer (Biolegend) to lyse the red blood cells. Remaining cells were washed and incubated with a cocktail of fluorochrome-conjugated antibodies (Cd4-PE-Cy7 (#552775) and Cd62L-FITC (#561917) from BD Pharmingen; Cd3e-PE (#12–0031), Cd8a-eFluor 450 (#48–0081) and Cd44-APC (#17–0441) from eBioscience.), incubated with propidium iodide for the detection of dead cells and analysed using the FACSCanto II analyser (BD Biosciences). The following T cell subsets were quantified: Cd3⁺, Cd8⁺, Cd44^high cytotoxic memory T cells; Cd3⁺, Cd8⁺, Cd44^low, Cd62L^high cytotoxic naïve T cells, Cd3⁺, Cd4⁺, Cd44^high helper memory T cells and Cd3⁺, Cd4⁺, Cd44^low, Cd62L^high helper naïve T cells.

Chemotaxis was determined with modified Migratest™ (Orpegen Pharma, Germany). Shortly, CSF was used as the medium contained unknown chemotaxis factors. Freshly isolated HC neutrophils were incubated in upper chamber (cell cultured insert with 3.0 µm ϕ) immersed in CSF (low chamber) for 30 min in 37°C. Neutrophils that have migrated through cell culture insert toward CSF or supernatant were labeled with propidium iodate, CD62L-FITC (fluorescein isothiocyanate) (BD) and analyzed within 30 min using flow cytometry (BD LSRII, FACSDiva™). The number of neutrophils was normalized using tubes containing constant number of microbeads (Trucount™, BD). As a positive chemotaxis control, N-formyl-methionyl-leucyl-phenylalanine (2 × 10⁻⁶ M) was used. PBS was used as a negative control.

All standard chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, United States) unless otherwise mentioned. ELISA antibodies, recombinant cytokines, and fluorochrome tagged antibodies for flow cytometry: F4/80-APC, CD11b-PerCP-Cy5.5, CD40-PE-Cy5, CD86-PE, MHC-II-PerCPefluor710, Annexin-FITC, CD4-PB, IL-17-PE, IFN-γ-PE-Cy7, CD62L-FITC, CD44-PerCP-Cy5.5, and CCR7-PECy7 are procured from BD Biosciences (San Diego, CA, United States). Antibodies for western blot analysis against: β-actin, goat anti-rabbit IgG-HRP, and donkey anti-mouse IgG-HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States); iNOS, pSTAT-1 (pY701), STAT-1, pSTAT-3 (pY705), STAT-3, pSTAT-6 (pY641) and STAT-6 antibodies were procured from Cell Signaling Technology (Danvers, MA, United States). Inhibitors used in the experiments such as STAT-1 inhibitor (fludarabine), Syk inhibitor (piceatannol) and iNOS inhibitor (NM, N^G-Monomethyl-L-arginine) are from Calbiochem (Billerica, MA, United States). Curdlan was purchased from InvivoGen (San Diego, CA, United States). All plastic-ware of tissue culture grade was procured from BD Biosciences (Bedford, MA, United States).

The patients’ PBMCs were purified by Ficoll-Hypaque (Celbio S.P.A., Italy) gradient separation of buffy coats of heparinized blood samples and analyzed by Fluorescence-activated cell sorting (FACS) analysis, as described in previous studies [5 (link), 6 (link)].
PBMC were stained with different pools of labeled monoclonal antibodies (mAbs) (CD4V450, CD45RAPE, CD62LFITC, CCR7PE-Cy7, Pharmingen;CD8PerCPCy 5.5, CD45ROAPC, CD3FITC, CD19FITC, CD14FITC, CD11cAPC, CD16PE, Rat IgG2aFITC, Mouse IgG1, Becton Dickinson, Italy; CD56 APC, Mouse IgG2a APC Immunotools, DE; CD15 PE ABCam, UK; CD25 PE, FoxP3 FITC, eBioscence, UK) and examined by a FACScalibur BD instrument. The fluorescent-minus-one and isotype control were included in each experiment in order to appropriately set the gates. Ki67 positive cells and T_regs were analyzed after intracellular immune-staining according to the manufacturer’s protocol (eBioscience). Cytofluorimetric analysis was carried out by using the FlowJo^® software. The mean fluorescence intensity (MFI) and percent of positive expression (%) of each marker were measured. Results were expressed as fold induction relative to baseline indicated as 1.

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).