Cd56 bv711

Manufactured by BioLegend

Sourced in United States

CD56-BV711 is a fluorochrome-conjugated antibody that binds to the CD56 cell surface antigen. CD56, also known as neural cell adhesion molecule (NCAM), is expressed on natural killer cells, a subset of T cells, and some tumor cells. The BV711 fluorochrome provides a bright signal for detection and analysis.

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5 protocols using cd56 bv711

Isolation and Characterization of Preleukemic Cells in Down Syndrome

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Viable frozen peripheral blood samples from 2 DS patients with DS-associated myeloid preleukemia were obtained from the biobank commission of the Princess Máxima Center for Pediatric Oncology. Mononuclear cells were stained with a cocktail of the following antibodies: CD3-BV650 (Biolegend, Clone UCHT1, 300467, 1:100), CD4-PerCP/Cy5.5 (Biolegend, Clone OKT4, 317427, 1:200), CD8-BV785 (Biolegend, Clone SK1, 344739, 1:100), CD19-BV421 (Biolegend Clone HCD14, 30224, 1:100) , CD14-AF700 (Biolegend, Clone HCD14, 325614, 1:100), CD56-BV711 (Biolegend, Clone HCD56, 318335, 1:50), CD34-APC (Biolegend, Clone 561, 343607, 1:50), CD38-PE (Biolegend, Clone HIT2, 303505, 1:50) , CD33-PE/Cy7 (Biolegend, Clone WM53, 303433, 1:100), CD117-PE-dazzle594 (Biolegend, Clone 104D2, 1:100), CD16-FITC (Biolegend, Clone 3G8, 302005, 1:100), CD20-FITC (Biolegend, Clone IVB201, 302303, 1:100). Bulk T-cells (CD3+/CD4+ and CD3+/CD8+) and preleukemic blast cells were sorted with the Astrios-EQ. Preleukemic blast cells were sorted according to the diagnostics flow data. Cell pellets were used for DNA isolation. Public data was used for the other 4 myeloid preleukemia samples^{11 (link)}.

Hasaart K.A., Manders F., van der Hoorn M.L., Verheul M., Poplonski T., Kuijk E., de Sousa Lopes S.M, & van Boxtel R. (2020). Mutation accumulation and developmental lineages in normal and Down syndrome human fetal haematopoiesis. Scientific Reports, 10, 12991.

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Multiparametric Analysis of CAR-NK Cells

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All samples were stained with anti-CD56-APC, anti-CD3-FITC (Biolegend) and 7AAD (BD Biosciences). Transgene expression was detected by flow cytometry on 7AAD⁻ CD56(-APC)⁺ CD3(-PE)⁻ cells (Biolegend). For NK-cell receptor detection, samples were stained with DAPI, CD56-BV711, CD16-BV786, NKp30-AF647, NKp44-PE, NKp46-BV421 (Biolegend), NKG2D-APC (BD Biosciences), and NKG2A-PE (Miltenyi Biotec). CD3-BV650 and CD19-APC-Cy7 (Biolegend) markers were used as a gating exclusion strategy for the NK cell staining. Receptor expression was assessed on DAPI⁻ CD56(-BV711)⁺ CD3(-BV650)⁻ cells. To detect CAR-expression, cells were incubated with 2 μl Siglec2(CD22)-Fc chimera (50 mg/ml, R&D) for 30 min at 4°C, washed and stained with anti-Fc-PE (Jackson Immune).

Colamartino A.B., Lemieux W., Bifsha P., Nicoletti S., Chakravarti N., Sanz J., Roméro H., Selleri S., Béland K., Guiot M., Tremblay-Laganière C., Dicaire R., Barreiro L., Lee D.A., Verhoeyen E, & Haddad E. (2019). Efficient and Robust NK-Cell Transduction With Baboon Envelope Pseudotyped Lentivector. Frontiers in Immunology, 10, 2873.

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Profiling NK Cell Subsets and Receptors

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In order to analyze NK cell subsets and their receptor expression profile, PBMCs were labeled with anti-CD3-BV785, -CD16-BV570, -CD38-BV510, -CD56-BV711, -CD57-FITC, -NKG2A-PE, -NKG2D-PE/Cy7, -NKp30-BV421, -NKp44-PE/Cy7 and -NKp46-AlexaFluor700 mAbs (all from Biolegend, San Jose, CA, USA) for 20 minutes at room temperature in the dark. The data were collected and analyzed with NovoCyte flow cytometer with NovoExpress operating system software (Agilent Technologies, USA).

Gelmez M.Y., Oktelik F.B., Tahrali I., Yilmaz V., Kucuksezer U.C., Akdeniz N., Cetin E.A., Kose M., Cinar C., Oguz F.S., Besisik S., Koksalan K., Ozdemir O., Senkal N., Gul A., Tuzun E, & Deniz G. (2022). Immune modulation as a consequence of SARS-CoV-2 infection. Frontiers in Immunology, 13, 954391.

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Profiling Interferon Signaling in Down Syndrome

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Phospho-STAT1 staining: Whole blood from individuals with DS and healthy controls were subject to Ficoll gradient to collect the mononuclear cell layer (PBMC) and frozen. For direct stimulation, thawed PBMCs were incubated in complete RPMI overnight, washed, stained with live-dead in PBS for 10 min at 37°C, and finally were stimulated for 15 min with indicated amounts of IFNα2b. For staining, cells were washed 2 times with 0.5% BSA and surface-stained on ice for 30 minutes (CD16-AF647, CD14-AF488, CD3-BV510, CD19-AF700, CD56-BV711, all from Biolegend) followed by fixation/permeabilization in 90% ice-cold methanol and staining with PE anti-phospho-STAT1 Y701 (1:25, BD).
IFNAR1 and IFNAR2 staining: hTERT-immortalized fibroblasts were scraped off in 5mM EDTA, washed, and stained with live-dead in PBS for 30 min on ice. They were then washed and stained with anti-IFNAR1 (clone AA3, courtesy of Sandra Pellegrini in Supplemental Fig 1A and clone MARI-5A3 from Millipore Sigma for Supplemental Fig 3A) or anti-IFNAR2 (PBL) for 2 hours on ice. The cells were washed and stained with biotin-conjugated rat anti-mouse IgG (H+L) (Thermo Fisher) for 40 min on ice, followed by PE-conjugated Streptavidin for 10 min on ice.
Flow cytometry was acquired on a Cytek Aurora or BD LSR Fortessa, and data were analyzed with Cytobank (https://www.cytobank.org/).

Malle L., Martin-Fernandez M., Buta S., Richardson A., Bush D, & Bogunovic D. (2022). Excessive Negative Regulation of Type I Interferon Disrupts Viral Control in Individuals with Down Syndrome. Immunity, 55(11), 2074-2084.e5.

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CFSE-based Lymphocyte Proliferation Assay

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To evaluate the proliferative responses of lymphocytes to C-Vx, carboxyfluorescein succinimidyl ester (CFSE) dilution method was used which is based on labelling cells with CFSE and evaluating the fluorescence halved by each cell division. PBMCs (up to 2 × 10⁷), freshly isolated and suspended in RPMI-1640 medium (Gibco, Paisley, UK), were stained with 1 μl of 5 mM CFSE solution (Thermo Fisher Scientific, USA) and incubated for 6 min at 4°C. Following washing with PBS, PBMCs were cultured for 120 h at 37°C with or without C-Vx (Miracle Labs PHArmaceutical Industry-Turkey) together with the absence or presence of 5 µl/mL PHA (Thermo Fisher, USA). Following cell culture, supernatants were collected and stored at −20°C for further analysis. PBMCs were harvested from the respective wells (US: unstimulated, PHA: phytohemagglutinin-stimulated, C-Vx, and PHA + C-Vx) into separate tubes for cell surface staining with anti-human-CD3-BV785, -CD4-PE-Cy7, -CD8-APC/Cy7, -CD16-BV570, and -CD56-BV711 (all from Biolegend, USA) mAbs. After the incubation, stained cells were washed with PBS and analyzed by flow cytometry.

Tahrali I., Akdeniz N., Yilmaz V., Kucuksezer U.C., Oktelik F.B., Ozdemir O., Cetin-Aktas E., Ogutmen Y., Ergen A., Abaci N., Tuzun E., Oncul O, & Deniz G. (2022). The modulatory action of C-Vx substance on the immune system in COVID-19. Emerging Microbes & Infections, 11(1), 2698-2710.

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