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Anti cd56

Manufactured by Miltenyi Biotec
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Sourced in United States, Germany

Anti-CD56 is a monoclonal antibody that binds to the CD56 surface antigen. CD56 is a cell adhesion molecule expressed on the surface of natural killer cells, a subset of T cells, and some other cell types. The primary function of Anti-CD56 is to detect and identify cells expressing CD56.

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Lab products found in correlation

Anti cd3, by Miltenyi Biotec (6 mentions) Anti cd16, by Miltenyi Biotec (4 mentions) Anti cd15, by Miltenyi Biotec (3 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Anti cd19 microbeads, by Miltenyi Biotec (2 mentions) Anti cd31, by Miltenyi Biotec (2 mentions) Anti cd14, by Miltenyi Biotec (2 mentions) Anti cd19, by Miltenyi Biotec (2 mentions) Macsquant analyzer 10, by Miltenyi Biotec (2 mentions) Anti cd45, by BD (2 mentions) Human b cell isolation kit 2, by Miltenyi Biotec (1 mentions) Anti cd14 microbeads, by Miltenyi Biotec (1 mentions) Bt474, by American Type Culture Collection (1 mentions) Hcc1806, by American Type Culture Collection (1 mentions) Dako qifikit, by Agilent Technologies (1 mentions) Pe conjugated anti cd3, by Miltenyi Biotec (1 mentions) Anti cd69, by Miltenyi Biotec (1 mentions) Mda mb 231, by American Type Culture Collection (1 mentions) Skbr3, by American Type Culture Collection (1 mentions) Anti cd14 61d3, by Thermo Fisher Scientific (1 mentions)

9 protocols using anti cd56

1

Isolation and Purification of PBMC Subsets

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Human PBMCs were isolated using Ficoll-Hypaque® density gradient centrifugation of heparinized venous blood from donors. The PBMCs were subjected to sequential isolation of T cells, NK cells and monocytes using anti-CD3, anti-CD56 and anti-CD14 microbeads according to manufacturer’s guidelines (Miltenyi Biotec Inc., San Diego, CA). Then the rest of the cells were further separated using a human B cell isolation kit II purchased from Miltenyi Biotec Inc. to obtain B cells and dendritic cells. In some experiments, we used antigen presenting cells (APCs, i.e. a mix of B cells, dendritic cells and monocytes) isolated from PBMCs by depletion of T cells and NK cells.
Bin L., Li X., Richers B., Streib J.E., Hu J.W., Taylor P, & Leung D.Y. (2018). ANKRD1 regulates innate immune responses against herpes simplex virus 1: A potential role in eczema herpeticum. The Journal of allergy and clinical immunology, 141(6), 2085-2093.e1.
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2

Mesothelin Expression in Cancer Cell Lines

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BT-474 (ATCC HTB-20), HCC1806 (ATCC® CRL-2335™), SK-BR-3 (ATCC® HTB-30™) and MDA-MB-231 (ATCC® HTB-26™) were purchased from ATCC. Ovarian cancer cells line, A1847 and human FcγRIIIA transfected Jurkat lymphoma T cells (Jurkat-huFcγRIIIA cells) were a gift of Dr N. Scholler, (SRI International, Ca USA) and Pr. Eric Vivier (Marseille, France), respectively, and were not authenticated. NK cells were isolated from human PBMCs as previously described (21 (link)). NK cells purity and activation status were determined by flow cytometry (MACSQuant® analyser (Miltenyi Biotec) after staining with PE-conjugated anti-CD3, anti-CD56, anti-CD16, anti-CD69, and anti-CD107 (Miltenyi Biotec). Mesothelin binding capacity of tumor cell lines was quantified by DAKO QIFIKIT (DAKO Cytomation), according to manufacturer's protocol using anti-human mesothelin monoclonal antibody K1 (GTX23362, GeneTex) as primary antibody. MSLN quantity was expressed as specific antibody-binding capacity units after substracting background from isotype control.
Del Bano J., Florès-Florès R., Josselin E., Goubard A., Ganier L., Castellano R., Chames P., Baty D, & Kerfelec B. (2019). A Bispecific Antibody-Based Approach for Targeting Mesothelin in Triple Negative Breast Cancer. Frontiers in Immunology, 10, 1593.
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3

Isolation and Sorting of Monocyte Subsets

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PBMCs were obtained by Ficoll density gradient centrifugation and were then depleted of granulocytes and lymphocytes using anti-CD15, anti-CD56, anti-CD3, and anti-CD19 microbeads (Miltenyi Biotec). The enriched monocyte fraction was then labeled with anti-CD14 [61D3] (#48-0149-42; eBioscience), anti-CD16 [VEP13] (#130-098-099; Miltenyi Biotec) and anti-CD56 [NCAM16.2] (#340363; BD Biosciences), for sorting by fluorescence-activated cell sorting (FACS) into the three monocyte subsets. For FACS, we first gated on single cells and then gated on the enriched monocytes population. We then excluded the CD14−CD56+ NK cells, as they also express CD16 (Supp Fig. S1). The remaining cells were gated into classical (CL; CD14high/CD16−), intermediate (ITM; CD14high/CD16+) and non-classical (NC; CD14low/CD16+) subsets (Fig. 1a).
Ong S.M., Hadadi E., Dang T.M., Yeap W.H., Tan C.T., Ng T.P., Larbi A, & Wong S.C. (2018). The pro-inflammatory phenotype of the human non-classical monocyte subset is attributed to senescence. Cell Death & Disease, 9(3), 266.
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4

Phenotypic Characterization of ADSCs

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Cultures of PF ADSCs and BC ADSCs at different passages (lower than passage 4) were phenotypically characterized following reference guidelines [32 (link), 33 (link)]. ADSCs obtained from PF- or BC-bearing patients were detached with 0.05% trypsin/EDTA (Thermo Fisher), washed with PBS, and 100000 cells were resuspended in 250 μL of PBS without Ca2+ and Mg+ (Euroclone, Pero, Italy) and incubated with antibodies directed against specific surface markers. Cells were incubated on ice for 30 minutes with antibodies anti-CD44 (BD Biosciences, San Jose, CA), anti-CD90 (Millipore, Massachusetts, USA), anti-CD34 (Miltenyi Biotec, Calderara di Reno, BO, Italy), anti-CD45 (BD Biosciences), anti-CD146 (Biocytex, USA), anti-CD31 (Miltenyi Biotec), anti-CD56 (Miltenyi Biotec), anti-CD105 (Serotec, Bio-Rad, Segrate, MI, Italy), anti-CD144 (R&D Systems, Minneapolis, MN, USA), anti-CD166 (BD Biosciences), anti-CD133/2 (Miltenyi Biotec), anti-CD73 (BD Biosciences), and anti-vascular endothelial growth factor 2 (VEGFR2; R&D Systems). Cells were pelleted, washed, and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes. Fluorescence-activated cell sorting (FACS) analysis was performed on a FACSVerse flow cytometer (BD Biosciences), equipped with the Cell Sweet software for data analysis.
Rey F., Lesma E., Massihnia D., Ciusani E., Nava S., Vasco C., Al Haj G., Ghilardi G., Opocher E., Gorio A., Carelli S, & Di Giulio A.M. (2019). Adipose-Derived Stem Cells from Fat Tissue of Breast Cancer Microenvironment Present Altered Adipogenic Differentiation Capabilities. Stem Cells International, 2019, 1480314.
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5

Comprehensive T-cell Phenotyping by Flow Cytometry

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Cellular composition, CD4/CD8 ratio and CAR-expression was determined by flow cytometric analysis after staining with one or several of the following antibodies and reagents: anti-CD45, anti-CD4, anti-CD3, anti-CD16, anti-CD56, anti-CD19, anti-CD14, anti-CD8, anti-CD45RO, anti-CD95, anti-CD62L and 7-AAD (all Miltenyi Biotec). CAR-modified T cells were identified using the EGFRt marker and staining with an anti-EGFRt antibody (clone: C225, ImClone Systems) conjugated in-house to AlexaFluor 647. Data were acquired on a MACSQuant Analyzer 10 and analyzed using MACSQuantify software (both Miltenyi Biotec).
Lock D., Monjezi R., Brandes C., Bates S., Lennartz S., Teppert K., Gehrke L., Karasakalidou-Seidt R., Lukic T., Schmeer M., Schleef M., Werchau N., Eyrich M., Assenmacher M., Kaiser A., Prommersberger S., Schaser T, & Hudecek M. (2022). Automated, scaled, transposon-based production of CAR T cells. Journal for Immunotherapy of Cancer, 10(9), e005189.
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6

Immunophenotypic Characterization of B Cell Subsets and Recent Thymic Emigrants

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Immunophenotype was performed on heparinized peripheral blood samples obtained from each patient by means of flow cytometry. 100 μL of blood were stained with two multicolour antibody panels in order to evaluate different lymphocyte subsets, including B cell subpopulations (naïve follicular B cells, marginal zone B cells, switched memory B cells and transitional B cells) and recent thymic emigrants (RTE).
Panel for B cells analysis contained the following antibodies: anti-CD45, anti-CD19, anti-CD38 (Becton Dickinson), anti-CD27, anti-IgM, anti-IgG, anti-CD21, anti-CD10 and anti-IgD (Miltenyi Biotec).
Panel for RTE analysis contained the following antibodies: anti-CD45, anti-CD19, anti-CD4, anti-CD8 (Becton Dickinson), anti-CD3, anti-CD45RA, anti-CD31, anti-CD16 and anti-CD56 (Miltenyi Biotec).
Samples were acquired with MACSQuant Analyzer 10 (Miltenyi Biotec) and analyzed with FlowLogic software (version 7.2.1, Inivai Technologies).
Girardelli M., Valencic E., Moressa V., Margagliotta R., Tesser A., Pastore S., Spadola O., Athanasakis E., Severini G.M., Taddio A, & Tommasini A. (2021). Genetic and immunologic findings in children with recurrent aphthous stomatitis with systemic inflammation. Pediatric Rheumatology Online Journal, 19, 70.
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7

Monocyte Subset Isolation and Analysis

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PBMCs were depleted of granulocytes and lymphocytes using anti-CD15, anti-CD56, anti-CD3, and anti-CD19 microbeads (Miltenyi Biotec). The enriched monocyte fraction was labeled with anti-CD14, anti-CD16, and anti-CD56 for fluorescence-activated cell sorting (FACS) into the three monocyte subsets. CD14−CD56+ NK cells were excluded, as they also express CD16 (Figure 2Cb). The remaining cells were gated into CL (CD14high/CD16−), ITM (CD14high/CD16+), and NC (CD14low/CD16+) subsets (Figure 2Cd).
Ong S.M., Teng K., Newell E., Chen H., Chen J., Loy T., Yeo T.W., Fink K, & Wong S.C. (2019). A Novel, Five-Marker Alternative to CD16–CD14 Gating to Identify the Three Human Monocyte Subsets. Frontiers in Immunology, 10, 1761.
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8

Quantifying Donor Leukocyte Composition

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Flow cytometry was used to quantify the distribution of different donor leukocytes in both the graft and perfusate compartments. Isolated cells were washed and stained with Zombie viability dye (Biolegend, San Diego, CA) in phosphate‐buffered saline, treated with Human Fc Receptor Blocking Solution (Biolegend), and then stained with the following markers prior to fixing with 2% formaldehyde (Fisher Scientific): anti‐CD3 (Biolegend), anti‐CD4 (Biolegend), anti‐CD8 (Biolegend), anti‐CD14 (Biolegend), anti‐CD16 (Biolegend), anti‐CD19 (Biolegend), anti‐CD45 (Invitrogen, Waltham, MA), anti‐CD56 (Miltenyl Biotec, Bergisch Gladbach, Germany), anti‐CD66b (Biolegend) (Supplemental Table 2). Data were acquired on an Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA) and analyzed using FlowJo Software (Version 10; FlowJo LLC, Ashland, OR) (Supplemental Figure 2).
Lee A.C., Edobor A., Wigakumar T., Lysandrou M., Johnston L.K., McMullen P., Mirle V., Diaz A., Piech R., Rose R., Jendrisak M., di Sabato D., Shanmugarajah K., Fung J., Donington J, & Madariaga M.L. (2022). Donor leukocyte trafficking during human ex vivo lung perfusion. Clinical Transplantation, 36(7), e14670.
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9

Multicolor Flow Cytometry and Magnetic Sorting

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The antibodies used for flow cytometry were anti-CD16 (clone 3G8), anti-CD14 (clone 61D3) (Biolegend, San Diego, CA). The antibodies used for magnetic sorting were anti-CD16, anti-CD14, anti-CD19, anti-CD3, anti-CD56 and anti-CD15 (Miltenyi Biotec, Bergisch Gladbach, Germany). The antibodies used for Western blotting were anti-RelA/nuclear factor-jΒ (mouse monoclonal, clone 532301) (Cell Signaling, Minneapolis, MN) and anti-GAPDH (rabbit polyclonal, IgG) (Imgenex, San Diego, CA).
Dang T.M., Wong W.C., Ong S.M., Li P., Lum J., Chen J., Poidinger M., Zolezzi F, & Wong S.C. (2015). MicroRNA expression profiling of human blood monocyte subsets highlights functional differences. Immunology, 145(3).
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