UCB units were provided from StemCyte under IRB-approved protocols. All donors provided written informed consent, which followed the ethical guidelines of the Declaration of Helsinki. NK cells were isolated by using the RosetteSepTM human NK cell enrichment cocktail (Cat# 15065, StemCell Technologies) and Ficoll-Paque (Cat# 17144003, Cytiva). The purity of primary NK cells was confirmed with flow cytometry using anti-CD56 (Cat# IM2474U, Beckman Coulter; 1:20 dilution) and anti-CD3 (Cat# 130-113-134, Miltenyi Biotec; 1:50 dilution) antibodies. Frozen UCB NK cells were thawed and expanded with irradiated K562 feeder cells expressing membrane-bound IL-21 and 4-1BBL (APC K562) in the presence of recombinant human IL-2 (50 IU/ml; NIH) in Stem Cell Growth Medium (SCGM) (Cat# 20802-0500, CellGenix). Expanded NK cells were transduced with retrovirus on day 5 in RetroNectin (Cat# T202, Takara Bio)-coated plates, according to the manufacturer’s protocol. On day 8, NK cells were co-cultured with irradiated APC K562 cells for an additional 7 days prior to being harvested for in vitro analysis or frozen (liquid nitrogen) for in vitro and in vivo studies. All NK cells used in our study did not undergo further purification, except where specifically indicated. Information on flow antibodies was presented in Supplementary Table 1 .
Anti cd56
Manufactured by Beckman Coulter
Sourced in United States
Anti-CD56 is a monoclonal antibody that recognizes the CD56 antigen, also known as neural cell adhesion molecule (NCAM). CD56 is expressed on the surface of natural killer cells, a type of lymphocyte involved in the immune response. The function of Anti-CD56 is to detect and identify cells expressing the CD56 antigen.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3, by Beckman Coulter (4 mentions)
Rosettesep human nk cell enrichment cocktail, by STEMCELL (2 mentions)
Anti cd3, by Miltenyi Biotec (2 mentions)
Anti cd8, by Beckman Coulter (2 mentions)
Anti cd45, by Beckman Coulter (2 mentions)
Anti cd83, by Beckman Coulter (2 mentions)
Fc500, by Beckman Coulter (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti cd86, by BD (2 mentions)
Anti ccr7, by R&D Systems (2 mentions)
Retronectin, by Takara Bio (1 mentions)
Ficoll paque, by Cytiva (1 mentions)
Cd163, by R&D Systems (1 mentions)
Anti cd19, by Beckman Coulter (1 mentions)
Facsdiva software, by BD (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Anti cd4, by Beckman Coulter (1 mentions)
Anti hla dr, by Beckman Coulter (1 mentions)
Anti tcr vβ11, by Beckman Coulter (1 mentions)
Epics xl mcl, by Beckman Coulter (1 mentions)
10 protocols using anti cd56
1
Expanding and Transducing Natural Killer Cells from Umbilical Cord Blood
2
Phenotyping of Mononuclear Cells
3
Multiparametric Flow Cytometry Analysis
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Surface phenotypes were determined using an Epics XL MCL (Beckman Coulter, Brea, CA, USA). The following monoclonal antibodies (mAbs) were purchased from Beckman Coulter: anti‐CD3, anti‐CD4, anti‐CD8, anti‐Vγ9TCR, anti‐CD14, anti‐CD25, anti‐CD45, anti‐CD54, anti‐CD56, anti‐HLA‐DR, anti‐CD40, anti‐CD80, anti‐CD86, anti‐CD11c, anti‐CD36, mouse immunoglobulin (Ig)G1, mouse IgG2 and mouse IgG2b mAbs. Anti‐HLA‐class 1 and anti‐CCR7 mAbs were purchased from Beckton Dickinson (San Jose, CA, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Anti‐TCR Vα24TCR and anti‐TCR Vβ11 mAbs were purchased from Beckman Coulter (Villepinte, France). Anti‐human CD273 (PD‐L2) and CD274 (PD‐L1) mAbs were purchased form eBioscience (San Diego, CA, USA). Anti‐human CD152 (CTLA‐4) and CD279 (PD‐1) mAbs were purchased from BioLegend (San Diego, CA, USA). Anti‐FoxP3 mAb for intracellular staining was purchased from BD Biosciences (Tokyo, Japan). All mAbs were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), antigen‐presenting cells (APC), extracellular domain (ECD), proprotein convertase (PC)5 or PC7. Z was purchased from Novartis Pharmaceuticals (Basel, Switzerland) and G from Funakoshi Co. Ltd (Tokyo, Japan).
4
Lymphocyte Subset Profiling by Flow Cytometry
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Cell surface markers of peripheral blood mononuclear cells were measured by flow cytometry (NAVIOS; Beckman Coulter) with immunofluorescent staining using anti-CD3, anti-CD4, anti CD8, anti-CD19, anti-CD16, and anti-CD56 antibodies (Beckman Coulter). T-cell receptor (TCR) v-β expression was determined as directed by the manufacturer (Beta Mark TCR v-β Repertoire Kit; Beckman Coulter). Reference range for lymphocyte subsets in adults were taken from Apoil et al. (15 (link))
5
Multiparametric Flow Cytometry Analysis
6
Isolation and Characterization of Primary NK Cells
7
Comprehensive Multiparametric Flow Cytometry Analysis
8
NK Cell-Mediated Cytotoxicity Assay
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mACE2-CAR_sIL15 NK and control NK cells were co-cultured with A549 or A549-spike cells at an E/T ratio of 4:1 for 4 h in a 96-well U-bottom plate. Anti-CD107a monoclonal antibody (mAb) (Cat# 563869, BD; 1:200 dilution) and GolgiPlug (1:1000 dilution) (Cat# 555029, BD) were added to cultures at the start of incubation. Cells were stained with anti-CD56 (Cat# IM2474U, Beckman Coulter; 1:20 dilution), anti-LNGFR (Cat# 557196, BD; 1:20 dilution) or anti-EGFR (Cat# 352904, BioLegend; 1:50 dilution) mAbs and then stained intracellularly with IFN-γ (Cat# 563563, BD; 1:20 dilution) and TNF-α (Cat# 557647, BD; 1:20 dilution) mAbs. The stained cells were analyzed by flow cytometry, and the data were analyzed with FlowJo software.
9
Phenotypic Analysis of NK Cells
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The Monoclonal antibodies (mABs) used for phenotypic analysis included Fluorescein isothiocyanate (FITC)-, Phycoerythrin (PE)-, Phycoerythrin Cyanine 5.5 and allophycocyanin (APC)-labeled anti-CD45, anti-CD3, anti-CD56, NKp30, NKp44 (Beckman Coulter, Brea, CA, USA), anti-CD16, NKp46, NKG2A (R&D System), anti-CD94, anti-CD69, anti-CD18, anti-CD49, anti-CD62L, anti-CD38 and CXCR4 (BD Pharmingen, San Jose, CA, USA). Phenotype evaluation was performed by direct immunofluorescence according to previously reported methods [29 (link)]. NK cells recovered from immunodeficient NOD/SCID gamma (NSG) mice were stained with anti-mouse CD45-APC-Cy7, anti-human CD45-PE-Cy7, CD3-PerCP-Cy5.5, CD56-FITC (Biolegend, San Diego, CA, USA) and CD94-APC (BD).
10
Comprehensive Lymphocyte Subset Analysis
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The leukapheresis products were suspended in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum and were analyzed using flow cytometry. Eight-colour analyses were performed for the identification of the lymphocyte subsets with the following monoclonal antibodies: CD2, CD3, CD4, CD5, CD7, CD16, CD25, CD56, CD45, CD45RA, CD45RO, CD9, CD127, CCR7, and HLA-DR antigens. All antibodies were purchased from BD Biosciences (San José, CA, USA) except for anti-CD8, anti-CD56, and anti-CD127 which were purchased from Beckman Coulter (Brea, CA, USA); anti-CCR7 was purchased from R&D systems (MN, USA) and anti HLA-DR from Biolegend (San Diego, CA, USA). The Blood Dendritic Cell Enumeration Kit (Miltenyi Biotech GmbH, Bergish Gladbach, Germany) was used according to the supplier's protocol to determine plasmacytoid dendritic cells, type 1 and type 2 myeloid dendritic cells. Flow cytometry analysis was performed on the LSR II instrument (BD Biosciences). Data analysis was performed using the Flow-Jo software (Tree Star, Ashland, OR, USA).
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