Apc anti human cd56

Manufactured by BioLegend

Sourced in United States

APC anti-human CD56 is a fluorochrome-conjugated antibody that binds to the CD56 cell surface antigen, which is expressed on natural killer (NK) cells and a subset of T cells. This antibody can be used for the identification and enumeration of CD56-positive cells in flow cytometric analyses.

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7 protocols using apc anti human cd56

IL-2 Production in Immune Cells of SLE Patients

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Peripheral blood mononuclear cells (PBMCs) in SLE patients and healthy donors were analyzed by flow cytometry (FACSAria II; BD Biosciences). To analyze IL-2 production, PBMCs were stimulated and incubated for 4 h with 50 ng/mL phorbol myristate acetate, 1

μ

g/mL ionomycin (both from Sigma-Aldrich), and 1

μ

L/mL GolgiStop (BD Biosciences). After stimulation, the cell surface markers CD4, CD3, CD8, CD56, CD25 and CD127 were stained with the fluorescence labeled monoclonal antibodies. Next, the cells were fixed and permeabilised for 30 min at 4°C at dark with BD FACS fixation and permeabilisation buffer set (BD Biosciences). Then the cells were stained with PE labeled anti-human-IL-2 antibody. Proportion of IL-2 expressing cells in CD4⁺ effector T cells (defined as CD4⁺ CD8⁻ CD127^hi/low CD25^low/−), double-negative T cells (DNT cell, CD3⁺ CD4⁻ CD8⁻), NK cells (CD56⁺ CD3⁻) and NKT cells (CD56⁺ CD3⁺) was analyzed using FlowJo v10 software (Tree Star). Antibodies used for flow cytometry included APC-H7-anti-human CD3 (Biolegend), Percp-anti-human CD8 (Biolegend), FITC-anti-human CD4 (Biolegend), BV421-anti-human CD25 (Biolegend), BV605-anti-human CD127 (Biolegend), APC-anti-human CD56 (Biolegend) and PE-anti-human IL-2 (Biolegend).

Gao X., He J., Sun X, & Li F. (2022). Dynamically modeling the effective range of IL-2 dosage in the treatment of systemic lupus erythematosus. iScience, 25(9), 104911.

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Isolation and Characterization of ccRCC Cells

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The surgically isolated ccRCC cancerous tissues were used for the preparation of single-cell suspensions after digestion with collagenase IV (30 mg/mL, Gibco, #17104-019). To remove debris, the samples were filtered through a 70 μm cell strainer. After counting, 1 × 10⁶ cells were used for staining for each sample, and the cells were suspended with MACS buffer (Miltenyi Biotec, #130-091-221). After blocking the potential unspecific binding sites, the cells were performed surface and intracellular staining with FITC anti-human CD16 (Biolegend, #980112), APC anti-human CD56 (Biolegend, #981204), Alexa Fluor 405 anti-human GZMH (GBiosciences, #ITT2050-405), PE anti-human EGR1 (Invitrogen, # 12-9851-42), and anti-human/anti-mouse CAPG (Creative Biolabs, #CBMAB-C0844-FY). The CAPG antibody was conjugated with Alexa Fluor 700 (Invitrogen, #A20110). The dead cells were counterstained with fixable viability dye. The stained samples were resuspended with PBS/0.5%BSA/2 mM EDTA buffer and recorded on a flow cytometer (ThermalFisher Attune Nxt).

Liang Z., Nong F., Zhao J., Wei D., Tang Q., Song J, & Meng L. (2022). Heterogeneity in NK Cell Subpopulations May Be Involved in Kidney Cancer Metastasis. Journal of Immunology Research, 2022, 6378567.

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Multicolor Flow Cytometry Analysis

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Pembrolizumab and nivolumab (anti-PD-1) were obtained from Merck and Bristol-Myers Squibb. The following antibodies were used for flow cytometry analyses: FITC anti-human CD4 (BioLegend Cat#391503), PE anti-human CD45 (Biolegend, clone HI30), APC anti-human CD56 (Biolegend, clone 5.1H11), APC/Cyanine7 anti-human CD8a (Biolegend, San Diego, CA, USA, clone RPA-T8), PE/Cyanine7 anti-human CD3 (Biolegend, clone HIT3a), PerCP/Cyanine5.5 anti-human CD314 (NKG2D) (Biolegend, clone 1D11), FITC anti-human CD6 (Biolegend, clone BL-CD6), APC anti-human CD16 (Biolegend, clone 3G8), Pacific Blue anti-human/mouse Ki67 (Biolegend, clone 16A8), FITC anti-human/mouse Granzyme B (Biolegend, clone GB11) and APC anti-human Perforin (Biolegend, clone B-D48).

Gurrea-Rubio M., Wu Q., Amin M.A., Tsou P.S., Campbell P.L., Amarista C.E., Ikari Y., Brodie W.D., Mattichak M.N., Muraoka S., Randon P.M., Lind M.E., Ruth J.H., Mao-Draayer Y., Ding S., Shen X., Cooney L.A., Lin F, & Fox D.A. (2023). Activation of Cytotoxic Lymphocytes Through CD6 Enhances Killing of Cancer Cells. Research Square.

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Immunophenotyping and Cytokine Quantification

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APC anti-human CD56, FITC anti-human CD3, PE anti-human CD16, murine isotype controls (IgG1κ-PE, IgG1κ-FITC, IgG2a –APC) and human IFN-γ ELISA MAX™ Deluxe kit were purchased from BioLegend Inc. (San Diego, CA). The recombinant human IL-2 protein was obtained from PeproTech (Rehovot, Israel). Calcein-AM was purchased from Sigma-Aldrich (St, Louis, MO). CCK8 kit was purchased from YESAN (Shanghai, China).

Gong C., Ni Z., Yao C., Zhu X., Ni L., Wang L, & Zhu S. (2015). A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells. Biological Procedures Online, 17, 12.

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DNA Methylation Profiling of CD8+ T Cells in Lupus

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For DNA methylation studies, eight patients with lupus and eight healthy controls recruited from the University of Michigan rheumatology clinics were matched by age (±5 years), sex and ethnicity (online supplementary table 1). Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood via Ficoll-Paque PLUS density centrifugation (GE Healthcare Bio-Sciences). Total T cells were isolated using negative selection by magnetic bead separation with the Human Pan T cell Isolation II kit (Miltenyi Biotec). Flow cytometry sorting was used to isolate CD3+ CD56-TCRαβ+ CD8+ T cells using the following fluorophore-conjugated antibodies: APC anti-human CD56 (clone: HCD56), FITC anti-human CD8a (clone: RPA-T8), Pacific Blue anti-human CD3 (clone: HIT3a) and PE anti-human alpha/beta-TCR (clone: IP26) (BioLegend), as previously described.^{23 (link)} DNA was extracted from CD8+ T cells using the DNeasy Blood and Tissue kit (Qiagen) and bisulfite converted with the EZ DNA Methylation kit (Zymo Research) for DNA methylation analysis. It is worth noting that TCRαβ+CD8+T cells constituted ~99% of CD8+ T cells in the peripheral blood of patients with lupus or controls.

Miller S., Tsou P.S., Coit P., Gensterblum-Miller E., Renauer P., Rohraff D.M., Kilian N.C., Schonfeld M, & Sawalha A.H. (2019). Hypomethylation of STAT1 and HLA-DRB1 is associated with type-I interferon-dependent HLA-DRB1 expression in lupus CD8+ T cells. Annals of the rheumatic diseases, 78(4), 519-528.

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Monoclonal Antibody Characterization Assay

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UMCD6, a mouse anti-human monoclonal antibody that targets domain 1 of CD6, was created in our laboratory and was purified from mouse ascites and desalted by column chromatography using Protein G and dextran per manufacturer’s instructions (Thermo Fisher Scientific). Pembrolizumab and nivolumab (anti-PD-1) were obtained from Merck and Bristol-Myers Squibb. The following antibodies were used for flow cytometry analyses: FITC anti-human CD4 (BioLegend Cat#391503), PE anti-human CD45 (Biolegend, clone HI30), APC anti-human CD56 (Biolegend, clone 5.1H11), APC/Cyanine7 anti-human CD8a (Biolegend, San Diego, CA, USA, clone RPA-T8), PE/Cyanine7 anti-human CD3 (Biolegend, clone HIT3a), PerCP/Cyanine5.5 anti-human CD314 (NKG2D) (Biolegend, clone 1D11), FITC anti-human CD6 (Biolegend, clone BL-CD6), APC anti-human CD16 (Biolegend, clone 3G8), Pacific Blue anti-human/mouse Ki67 (Biolegend, clone 16A8), FITC anti-human/mouse Granzyme B (Biolegend, clone GB11) and APC anti-human Perforin (Biolegend, clone B-D48).

Gurrea-Rubio M., Wu Q., Amin M.A., Tsou P.S., Campbell P.L., Amarista C.I., Ikari Y., Brodie W.D., Mattichak M.N., Muraoka S., Randon P.M., Lind M.E., Ruth J.H., Mao-Draayer Y., Ding S., Shen X., Cooney L.A., Lin F, & Fox D.A. (2024). Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells. Cancer Immunology, Immunotherapy : CII, 73(2), 34.

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NK Cell Cytotoxicity Assay with Engineered Raji Cells

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Raji β2-microglobulin knockout and HHLA2 transfected Raji β2-microglobulin knockout cells were co-cultured in RPMI1640 medium supplemented with 10% FBS, 1% Gluta-Max and 1% Penicillin/Streptomycin with NK-92 MI cells for 3 hours at E:T ratios of 1:1, 2:1 and 3:1 in round-bottom 96-well plates at 37°C with 5% CO₂. Anti-KIR3DL3 (1G7) and mouse IgG2b isotype control (Bxcell; cat. #BE0086) were used at 10μg/ml. BV421 anti-human CD107a (Biolegend; cat. #328625), APC anti-human CD56 (Biolegend; cat. #362503), APC Mouse IgG1, κ Isotype control (Biolegend; cat. #400121) and BV421 Mouse IgG1, κ Isotype control (Biolegend; cat. #400157) were used at 1:100 dilution. Monensin Solution (1,000X) (Biolegend; cat 420701) was used at 1:1000 dilution and Cell Stimulation Cocktail, PMA/Ionomycin (Biolgend; cat. #423301) was used at 1:500 dilution. Subsequently the plate was put on ice and cells were analyzed for CD107a expression on CD56 gated cells using flow cytometry and FlowJo software.

Bhatt R.S., Berjis A., Konge J.C., Mahoney K.M., Klee A.N., Freeman S.S., Chen C.H., Jegede O.A., Catalano P.J., Pignon J.C., Sticco-Ivins M., Zhu B., Hua P., Soden J., Zhu J., McDermott D.F., Arulanandam A.R., Signoretti S, & Freeman G.J. (2020). KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1. Cancer immunology research, 9(2), 156-169.

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