Cd56 fitc

Manufactured by Thermo Fisher Scientific

Sourced in Germany

CD56-FITC is a fluorescently labeled antibody that binds to the CD56 antigen. CD56 is a cell surface glycoprotein expressed on natural killer cells, a subset of T cells, and some neuronal cells. The FITC (Fluorescein Isothiocyanate) label allows for the detection and identification of cells expressing CD56 using flow cytometry or other fluorescence-based techniques.

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5 protocols using cd56 fitc

Characterization of NK Cell Subsets

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NK cells were characterized as CD3 negative cells with a specific CD panel (CD56-FITC, CD16-PerCp-Cy5.5, CD107a-PE) (e-Bioscience, Frankfurt, Germany), anti-KIR2DL2-2DS2-2DL3/CD158b-PE (ThermoScientific, Erembodegem, BE) monoclonal antibodies. Samples were incubated with the moAbs for 30 min in ice and washed. The analysis was performed with FACS CantoII flow cytometer and FlowJo software (Becton Dickinson, San Jose, CA, USA), acquiring 10,000 events. Lymphocytes were identified according to the forward/side scatter profile and NK cells (CD3−/CD56+) were defined and gated within the lymphocyte gate. For the CD107a degranulation assay, after 1 h of incubation at 37 °C and 3 h of treatment with Golgi Stop solution (Becton Dickinson, San Jose, CA, USA), PBMCs were stained. CD158b levels were evaluated in the CD3−/CD56+/CD16+ gated cells. Cell viability was assessed by propidium iodide staining. Anti-isotype controls (Exbio, Praha, Czech Republic) were performed. Ten thousand events were acquired.

Bortolotti D., Gentili V., Bortoluzzi A., Govoni M., Schiuma G., Beltrami S., Rizzo S., Baldi E., Caselli E., Pugliatti M., Castellazzi M., Fernández M., Fainardi E, & Rizzo R. (2022). Herpesvirus Infections in KIR2DL2-Positive Multiple Sclerosis Patients: Mechanisms Triggering Autoimmunity. Microorganisms, 10(3), 494.

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Multiparameter Immune Cell Profiling

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CD3-percp-cy5.5 (Catalogue #45-0036-42), CD8-APC (Catalogue #17-0086-42), CD8-FITC (Catalogue #11-0086-42), CD14-APC (Catalogue #17-0149-42), CD56-FITC (Catalogue #4278380), CD16-PerCP-eFluor™710 (Catalogue #46-0168-42), CD11b-PerCP-eFluor™710 (Catalogue #46-0110-80), IFN-γ-PE (Catalogue #12-7319-42), TLR2-FITC (Catalogue #11-9922-41) and Mouse IgG1-PE (Catalogue #12-4714-81) were all bought from eBioscience. HLA-DR-PE (Catalogue #555812), IL-10-APC (Catalogue #554707), CXCR3-APC (Catalogue #550967) and IFN-γ-FITC (Catalogue #561053), Rat IgG2a-APC (Catalogue #554690) and mouse IgG-APC (Catalogue #5065947) were purchased from BD Biosciences. CD16-FITC (Catalogue #302006), HLA-DR-APC (Catalogue #307609), CD3-FITC (Catalogue #317306) and TLR4-APC (Catalogue #312815) were purchased from Biolegend. EP2-PE (Catalogue #10477) and Rabbit IgG-PE (Catalogue D5-1610) were purchased from Cayman Chemical.

Wang Y., Chen C., Qi J., Wu F., Guan J., Chen Z, & Zhu H. (2019). Altered PGE2-EP2 is associated with an excessive immune response in HBV-related acute-on-chronic liver failure. Journal of Translational Medicine, 17, 93.

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Flow Cytometry Analysis of Differentiated Cells

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For flow-cytometry analysis of surface markers, cells were harvested during the proliferation phase at day 20 of differentiation. Cells were dissociated with 0.25% trypsin-EDTA, washed with PBS, and resuspended in flow buffer (PBS with 5% FBS). Cells were incubated with the following conjugated antibodies at 0.25 μg/10⁶ cells: immunoglobulin G1-K isotype control-fluorescein isothiocyanate (FITC) (eBioscience 11-4714-41), CD56-FITC (eBioscience 11-0566-41), or CD29-FITC (eBioscience 11-0299-41). Cells were analyzed on a Sony SH800 flow cytometer.

Kwon J.B., Vankara A., Ettyreddy A.R., Bohning J.D, & Gersbach C.A. (2020). Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-Based Transcriptional Activators. Stem Cell Reports, 14(5), 755-769.

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Polychromatic Flow Cytometry Analysis

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The following fluorochrome-conjugated Abs were used for polychromatic flow cytometry analysis: CD3-Pacific Blue (UCHT1), CD4-Alexa700 (RPA-T4), CD45RA-APC-Cy7 (HI100), CCR6-PE (11A9), CCR7-PE-Cy7 (3D12), CD25-PE (M-A251), CD26-FITC (L272), CD127-AF647 (HIL-7R-M21), CD161-PE-Cy5 (DX12), IFNγ-Alexa 700 (B27), CCR5-PE (2D7), CXCR4-PE (12G5), and CD8-APC H7 (SK1) (BD Pharmingen), CD45RA-APC eFluor780 (HI100), CD56-FITC (MEM188), IL-17A-PE (eBio64DEC17), FoxP3-AF488 (PCH101), TNFα-Pacific Blue (Mab11), and IL-17A-eFluor660 (eBio64CAP17) (eBioscience), CD8-FITC (BW135/80), CD19-FITC (LT19) (Miltenyi), CCR7-PE (150503) (R&D) and CD31-BV605 (WM59) (Biolegend). Cell phenotype was analyzed by flow cytometry using the BD LSRII cytometer and BD Diva software. A viability staining Vivid (Invitrogen) was included in each staining cocktail to exclude dead cells from our analysis. FACS analysis was performed using the FlowJo software (^©Tree Star, Inc.). For multicolor analysis, all Abs were titrated for an optimal noise/signal ratio and Abs cocktails were validated by comparing single to multiple staining. Positivity gates were placed based on fluorescence minus one (FMO), as previously described [21 (link),101 ].

DaFonseca S., Niessl J., Pouvreau S., Wacleche V.S., Gosselin A., Cleret-Buhot A., Bernard N., Tremblay C., Jenabian M.A., Routy J.P, & Ancuta P. (2015). Impaired Th17 polarization of phenotypically naive CD4+ T-cells during chronic HIV-1 infection and potential restoration with early ART. Retrovirology, 12, 38.

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Multiparametric FACS Analysis of Immune Cells

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Single-cell suspension was obtained after filtration and centrifugation. The cells for FACS were stained with Fluorophore-conjugated antibodies containing CD45-eFluor (Cat# 69-0459-42, eBioscience™), CD3-FITC (Cat# 11-0037-42, eBioscience™), CD56-FITC (Cat# 11-0566-42, eBioscience™), CD19-FITC (Cat# 11-0199-42, eBioscience™), CD1c-APC (Cat# 331524, BioLegend), CD163-PE (Cat# 12-1639-42, eBioscience™), CD14-BV421(Cat# 563743, DB Biosciences) on ice for 30 min. After washing twice with FACS buffer, the cells were stained using 7-AAD Viability Staining Solution (Cat# 00-6993-50, eBioscience™) on ice 5 min. FACSAriaIII (BD Biosciences) was used for FACS. FACS data were collected using BD FACS Diva Software (version 8.0.2, BD), and data were analyzed with FlowJo software. Detailed information of antibody application and dilution is shown in Supplementary Table 4.

Huang X.Z., Pang M.J., Li J.Y., Chen H.Y., Sun J.X., Song Y.X., Ni H.J., Ye S.Y., Bai S., Li T.H., Wang X.Y., Lu J.Y., Yang J.J., Sun X., Mills J.C., Miao Z.F, & Wang Z.N. (2023). Single-cell sequencing of ascites fluid illustrates heterogeneity and therapy-induced evolution during gastric cancer peritoneal metastasis. Nature Communications, 14, 822.

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