Anti cd56 bv605

Manufactured by BioLegend

Anti-CD56-BV605 is a fluorochrome-conjugated antibody that binds to the CD56 cell surface antigen. CD56 is a neural cell adhesion molecule (NCAM) expressed on natural killer cells, a subset of T cells, and some types of neurons. The BV605 fluorochrome emits light in the violet spectrum upon excitation, allowing for detection and analysis of CD56-positive cells using flow cytometry or other applications.

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Anti-CD56 (17 citations) CD56-BV605 (2 citations)

5 protocols using anti cd56 bv605

Assessing NK Cell Cytotoxicity and Cytokine Production

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NK cell cytotoxicity and cytokine production was assessed on 15 patients at baseline and at the beginning of courses 3 and 6, and on 3 HDs. PBMCs were preincubated alone (negative control) or with K562 target (E:T ratio of 10:1) for 5 hours at 37°C, in the presence of anti-CD107a-PECF594 mAb, GolgiStop/monensin (BD Biosciences) and Brefeldin A. Cells were then stained with Live/Dead Aqua Viability Marker, anti-CD3-APC-Cy7, and anti-CD56-BV605 mAbs (both from Biolegend). After surface staining cells were lysed, fixed, and permeabilized. Cytokine production was detected by intracellular staining with anti-IFNγ-v450 and anti-TNFα- Alexa 700 mAb. Flow cytometry data were acquired on BD LSRFortessa and analyzed on FlowJo software.

Vitale C., Falchi L., ten Hacken E., Gao H., Shaim H., Van Roosbroeck K., Calin G., O'Brien S., Faderl S., Wang X., Wierda W.G., Rezvani K., Reuben J.M., Burger J.A., Keating M.J, & Ferrajoli A. (2016). Ofatumumab and Lenalidomide for Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia: Correlation between Responses and Immune Characteristics. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(10), 2359-2367.

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BCG-specific T cell Profiling

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PBMCs from four BCG-vaccinated healthy controls and P were stained with the CellTrace Far-Red Cell Proliferation Kit (Thermo Fisher Scientific) at a dilution of 1:25,000. Cells were washed and resuspended at a density of 2 x 10⁶ cells/mL in RPMI 10% human AB serum (Gemini). Cells were plated in 96-well U-bottomed plates and stimulated with BCG-lysate at a final concentration of 5 μg/mL or with tuberculin purified protein derivative (PPD) at a final concentration of 5 μg/mL (STATEN SERUM INSTITUT). Cells were cultured for 6 days and then harvested and stained with the Zombie NIR Viability kit (BioLegend) for 15 minutes. Cells were then surface-stained with FcBlock, anti-CD3-V450 (BD Biosciences), anti-CD4-BUV563 (BD Biosciences), anti-CD8-BUV737 (BD Biosciences), anti-Vδ2TCR-APC/Fire750 (BioLegend), anti-CD56-BV605 (BioLegend) and anti-CD20-BV785 (BioLegend) antibodies for 30 minutes. Cells were fixed with the FOXP3/Transcription Factor Buffer kit (Thermo Fisher Scientific) and stained by overnight incubation with anti-T-bet-PE/Cy7 (BioLegend), anti-IFN-γ-BV711 (BioLegend), anti-TNF-α-BV510 (BioLegend) and anti-human/mouse-Ki-67 (BioLegend) antibodies in Perm/Wash buffer. Data were acquired in a CyTek Aurora spectral flow cytometer.

Yang R., Mele F., Worley L., Langlais D., Rosain J., Benhsaien I., Elarabi H., Croft C.A., Doisne J.M., Zhang P., Weisshaar M., Jarrossay D., Latorre D., Shen Y., Han J., Ogishi M., Gruber C., Markle J., Ali F.A., Rahman M., Khan T., Seeleuthner Y., Kerner G., Husquin L.T., Maclsaac J.L., Jeljeli M., Errami A., Ailal F., Kobor M.S., Oleaga-Quintas C., Roynard M., Bourgey M., Baghdadi J.E., Boisson-Dupuis S., Puel A., Batteux F., Rozenberg F., Marr N., Pan-Hammarström Q., Bogunovic D., Quintana-Murci L., Carroll T., Ma C.S., Abel L., Bousfiha A., Santo J.P., Glimcher L.H., Gros P., Tangye S.G., Sallusto F., Bustamante J, & Casanova J.L. (2020). Human T-bet governs innate and innate-like adaptive IFN-γ immunity against mycobacteria. Cell, 183(7), 1826-1847.e31.

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Multiparameter Flow Cytometry of NK Cells

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Flow cytometry of NK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Staining of NK cells was performed with four panels, and the antibodies used were: anti-CD56-BV605, anti-NKG2D-APC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-4-1BB-PerCP-Cy5.5, anti-CD95-BV421, anti-Tim3-BV421, anti-TRAIL-PE, anti-CD122-PerCP-Cy5.5, anti-CD122-BV510, anti-CD95L-PE, anti-Ki67-BV421, anti-CD40L-BV510, anti-PD-1-PE-Cy7; all were obtained from BioLegend (San Diego, CA). Anti-CD16-APC-H7, PD-L1-PE-Cy7, and CD11a-FITC, were obtained from BD Biosciences (San Jose, CA). Anti-NKG2A-PE was from R&D Systems (Minneapolis, MN), and anti-CD158a-PerCP-Cy5.5 from eBioscience.

Jochems C., Tritsch S.R., Pellom S.T., Su Z., Soon-Shiong P., Wong H.C., Gulley J.L, & Schlom J. (2017). Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824). Oncotarget, 8(43), 75217-75231.

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Comprehensive Characterization of T-cell Subsets

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Freshly thawed PBMCs (1.0–1.5×10⁶ cells) were stained with the Zombie NIR Fixable Viability Kit (BioLegend, 1:2000) for 15 minutes at room temperature. Cells were then stained with the following panel for 30 minutes on ice: FcR blocking reagent (Miltenyi Biotec, 1:50), anti-CD3-Alexa Fluor 532 (eBioscience, Clone: UCHT1, Cat: 58–0038-42, 1:50), anti-CD4-BV750 (BD Biosciences, Clone: SK3, Cat: 566356, 1:800), anti-CD8a-BV510 (BioLegend, 1:200), anti-CD56-BV605 (BioLegend, Clone: 5.1H11, Cat: 362537, 1:100), anti-γδTCR-FITC (eBioscience, Clone: B1.1, Cat: 11–9959-41, 1:50), anti-Vδ1-VioBlue (Miltenyi Biotec, Clone: REA173, Cat: 130–120-583, 1:50), anti-Vδ2-APC/Fire 750 (BioLegend, Clone: B6, Cat: 331419, 1:100), anti-CD161-PE (BioLegend, Clone: HP-3G10, Cat: 339938, 1:100), anti-Vα7.2-BV711 (BioLegend, Clone: 3C10, Cat: 351731, 1:100), anti-Vα24-Jα18-PE/Cy7 (BioLegend, Clone: 6B11, Cat: 342912, 1:100), and anti-Vβ11-APC (Miltenyi Biotec, Clone: REA559, Cat: 130–125-508, 1:50) antibodies. The cells were fixed by incubation with 2% PFA in PBS for 15 minutes at room temperature in the dark. Cells were acquired with an Aurora Flow Cytometer (Cytek). Data were analyzed with FlowJo.

Ogishi M., Yang R., Aytekin C., Langlais D., Bourgey M., Khan T., Ali F.A., Rahman M., Delmonte O.M., Chrabieh M., Zhang P., Gruber C., Pelham S.J., Spaan A.N., Rosain J., Lei W.T., Drutman S., Hellmann M.D., Callahan M.K., Adamow M., Wong P., Wolchok J.D., Rao G., Ma C.S., Nakajima Y., Yaguchi T., Chamoto K., Williams S.C., Emile J.F., Rozenberg F., Glickman M.S., Rapaport F., Kerner G., Allington G., Tezcan I., Cagdas D., Hosnut F.O., Dogu F., Ikinciogullari A., Rao V.K., Kainulainen L., Béziat V., Bustamante J., Vilarinho S., Lifton R.P., Boisson B., Abel L., Bogunovic D., Marr N., Notarangelo L.D., Tangye S.G., Honjo T., Gros P., Boisson-Dupuis S, & Casanova J.L. (2021). Inherited PD-1 deficiency underlies tuberculosis and autoimmunity in a child. Nature medicine, 27(9), 1646-1654.

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Autologous NK Cell Activation Assay

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NK cells were isolated from cryopreserved PBMCs of autologous donors or individuals matched for HLA-Bw4/Bw6 and C1/C2 motifs to organoid donor, and stimulated as described above. WT and HLA-DPB1 KO organoids were expanded, stimulated with IFN-γ (200 U/mL, 72 hours) and dissociated to single cells. Prestimulated NK cells were resuspended in RPMI-1640/10% FBS at final concentration of 2×10⁵ cells/mL. Cholangiocyte organoids and NK cells were distributed to the respective conditions at a E:T ratio of 1:10 and anti-CD107a-BV785 (BioLegend) was added. Plate-bound anti-NKp44 and PBS only served as positive and negative controls. Cells were incubated for 5 hours at 37°C/5% CO₂, and subsequently stained with anti-CD3-BV510, anti-CD56-BV605, anti-CD16-FITC, anti-NKp44-AF647, anti-CD69-BV421 (all BioLegend) and LIVE/DEAD Fixable Near-IR Dead Cell Stain (Invitrogen). Cells were washed, fixed in 4% PFA and analysed at a BD LSRFortessa.

Zecher B.F., Ellinghaus D., Schloer S., Niehrs A., Padoan B., Baumdick M.E., Yuki Y., Martin M.P., Glow D., Schröder-Schwarz J., Niersch J., Brias S., Müller L.M., Habermann R., Kretschmer P., Früh T., Dänekas J., Wehmeyer M.H., Poch T., Sebode M., Ellinghaus E., Degenhardt F., Körner C., Hoelzemer A., Fehse B., Oldhafer K.J., Schumacher U., Sauter G., Carrington M., Franke A., Bunders M.J., Schramm C, & Altfeld M. (2023). HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells. Gut, 73(2), 325-337.

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