Anti cd56 pe

PBL subsets were isolated from cirrhotic patients or healthy individuals by flow cytometry. Three to five milliliters of peripheral blood was collected with lithium heparin. After washing with PBS, anti-CD8⁺-PE, anti-CD4⁺-FITC, anti-CD56⁺-PE, and anti-CD16⁺-FITC antibodies were added to the cell suspension (Ebioscience). The subsets of PBLs were enriched by flow cytometry. Enriched cell fractions were counted with a counting plate and the viability of cells was about 95% as measured by the Trypan Blue Staining. The purity of CD8⁺T cells, CD4⁺T cells, or NK cells was 99.4%, 99.7%, or 96.8%, respectively (data not shown).

Primary NK cells were purified from peripheral blood mononuclear cells (PBMCs) of healthy donors using the NK cells isolation kit (Miltenyi Biotec, 130-092-657) according to manufacturer's protocol. After isolation, the isolated cells were maintained in IL-2 containing NK cell media. The purity of isolated cells (CD56+CD3-) was confirmed in flow cytometric analysis by staining with anti-CD56-PE (e-Bioscience, 12-0267-41) and anti-CD3-Cy7 (BioLegend, 300429) antibodies.

We had two NK cell sources for the NK-cytotoxicity tests: NK 92 and primary NK cells. The established NK cell line NK92 was purchased from ATCC (CRL2407^TM). The primary NK cells were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy donors using NK cell isolation kit (Miltenyi Biotec, 130-092-657) according to manufacturer’s protocol. After the isolation, isolated cells were maintained in IL-2 containing NK cell media. The purity of isolated cells (CD56+ CD3−) was confirmed by flow cytometric analyses using anti-CD56-PE (e-Bioscience, 12-0267-41) and anti-CD3-Cy7 (BioLegend, 300429) antibodies.

Blood samples for cell population analysis were collected one day before conditioning chemotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples (10 mL) collected in EDTA-coated tubes by centrifugation in Ficoll-Paque Plus. PBMCs were processed immediately for analysis. Flow cytometry was used to evaluate the percentages of CD3⁺, CD4⁺CD161⁺, and CD8⁺CD161⁺ T cells; natural killer (NK) cells (CD16⁺CD56⁺); and myeloid-derived suppressor cells (MDSCs) [Lin⁻HLA-DR⁻CD11b⁺CD33⁺ (granulocytic) and HLA-DR⁻CD14⁺ (monocytic)]. Anti-CD3-allophycocyanin (APC), anti-CD4-fluorescein isothiocyanate (FITC), anti-CD8-phycoerythrin (PE), anti-CD161-PerCP-Cy5.5, anti-CD16-FITC, anti-CD56-PE, and anti-CD14-APC monoclonal antibodies (mAbs) were purchased from eBioscience (San Diego, CA, USA). Anti-Lineage cocktail 1 (Lin 1)-FITC, anti-HLA-DR-PerCP, rat anti-mouse CD11b-APC-Cy™7, and mouse anti-human CD33-V450 (BD BioSciences) mAbs were purchased from BD Biosciences (San Jose, CA). CD4⁺CD161⁺ and CD8⁺CD161⁺ T cells were gated on CD3⁺ cells and are expressed as percentages of lymphocytes. The frequencies of HLA-DR⁻Lin⁻CD11b⁺CD33⁺ and HLA-DR⁻CD14⁺ MDSCs are expressed as percentages of total PBMCs. Flow cytometry was performed using a FACS LSR Fortessa (BD Biosciences).

NK cell isolation kits (Miltenyi Biotec, Gladbach, Germany) were used to purify primary NK cells from peripheral blood mononuclear cells (PBMCs) of healthy people. The isolated cells were kept in IL-2 containing NK cell media and stained with anti-CD56-PE (e-Bioscience, CA, USA) and anti-CD3-Cy7 (BioLegend, CA, USA). Flow cytometry was employed to measure the purity of separated cells (CD56⁺CD3⁻) and the cells were cultured in α-minimum essential medium containing sodium bicarbonate (Sigma), IL-2 (100 units/mL), inositol (0.2 mM), 2-mercaptoethanol (0.1 mM), folic acid (0.02 mM), 12.5% horse serum and 12.5% FBS (Hyclone Laboratories, Logan, UT).