Cd16 bv650

Manufactured by BioLegend

Sourced in Germany

CD16-BV650 is a fluorochrome-conjugated antibody that binds to the CD16 cell surface antigen. CD16 is a low-affinity receptor for the Fc portion of IgG antibodies, expressed on natural killer cells, neutrophils, and macrophages. This product can be used in flow cytometry applications to identify and analyze CD16-positive cells.

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Anti-CD16 BV650 (2 citations)

5 protocols using cd16 bv650

Multiparametric Flow Cytometry Analysis

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Acquisitions were performed using a Beckman Coulter Cytoflex flow cytometer, and data were analyzed by the Kaluza software (Beckman Coulter, Milan, Italy). Cytoflex Daily QC Fluorospheres (Beckman Coulter) were used to calibrate the flow cytometer. The following monoclonal fluorochrome-conjugated antibodies were used for cell labeling of CD14⁺ cells from NKEV-monocyte cultures and PBMCs harvested from LPS-stimulated NKEV-PBMC cocultures: CD3-KO (Beckman Coulter), CD14-Alexa 750 (Beckman Coulter), CD16-BV650 (Biolegend), CD80-86-PE (Becton Dickinson), and HLA-DR-APC (Beckman Coulter).
The following antibodies were used for cell labeling of peripheral blood lymphocytes (PBL), NK cells and PBMCs harvested from CD3/CD28-stimulated or unstimulated NKEV-PBMC cocultures, in the presence or absence of TGFβ/IL-10: CFSE (CellTrace, Thermo Fisher Scientific), CD3-ECD (Beckman Coulter), CD4-Alexa700 (Beckman Coulter), CD8-BV605 (Biolegend), CD56-BV510 (Becton Dickinson), CD16-BV650 (Biolegend), PD-1-PC7 (Beckman Coulter), CD25-PercpCy5.5 (Becton Dickinson), HLA-DR-APC (Beckman Coulter), and CD14-Alexa750 (Beckman Coulter).
Samples were incubated with Fc blocking reagent (Miltenyi Biotec, Germany) for 10 min at room temperature before the addition of monoclonal antibodies for 40 min at 4°C. Thereafter, samples were washed and fixed.

Federici C., Shahaj E., Cecchetti S., Camerini S., Casella M., Iessi E., Camisaschi C., Paolino G., Calvieri S., Ferro S., Cova A., Squarcina P., Bertuccini L., Iosi F., Huber V, & Lugini L. (2020). Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors. Frontiers in Immunology, 11, 262.

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Multiparameter Flow Cytometry of NK Cells

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PBMC was stained using Zombie Live/dead fixable cell stain (Thermo Fisher Scientific, Waltham, MA, USA) prior to staining with specific antibodies. Antibodies used were CD3 BUV496 (BD Bioscience), CD19 PeCy7 (BD Bioscience), CD56 BB700 (BD Bioscience), CD16 BV650 (BioLegend), CD96 BV421 (BD Bioscience), TIGIT BUV395 (BD Bioscience), DNAM1 (CD226) BUV805 (BD Bioscience). PBMC was stained in Live/Dead cell stain for 15 min in PBS at RT, washed in FACS buffer (PBS + 2% FCS), and blocked in Human Fc block (BD Bioscience) for five minutes at RT. Cell staining was performed in FACS buffer for 30 min at 4 °C, followed by two washes in FACS buffer, and fixation in 2% paraformaldehyde (PFA, Electron Microscopy Sciences). Samples were acquired on a LSR Fortessa (BD Bioscience) flow cytometer and analysed using FlowJo software (BD Bioscience). Flow cytometry gating is shown in Additional file 1: Fig. S1. NK cells were defined as CD3-CD19-CD56 + viable single lymphocytes. NK cell subsets were defined as mature (CD16 + CD56mid), regulatory (CD16-CD56mid) and immature (CD16-CD56hi). Expression of DNAM1, TIGIT and CD96 was calculated using the geometric mean of the mature, regulatory and immature NK cell subsets.

Jake S.h.o.r.t.t., Galettis P., Cheah C.Y., Davis J., Ludford-Menting M., Link E.K., Martin J.H., Koldej R, & Ritchie D. (2023). A phase 1 clinical trial of the repurposable acetyllysine mimetic, n-methyl-2-pyrrolidone (NMP), in relapsed or refractory multiple myeloma. Clinical Epigenetics, 15, 15.

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Comprehensive Lymphocyte Immunophenotyping

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The following antibodies were used for phenotyping and functional evaluation of lymphocytes in this study: CD107a-BV421 (Clone H4A3, Biolegend), CD4-PECy5.5 (Clone S3.5, Invitrogen), CD8-BV570 (Clone RPA-T8, Biolegend), CD14-BV650 (Clone M5E2, Biolegend), CD20-BV650 (Clone 2H7, Biolegend), CD16-BV650 (Clone 3G8, Biolegend), CD28-ECD (Clone CD28.2, Beckman Coulter), CD95-PECy5 (Clone Dx2, BD Biosciences), CD38-PE (Clone OKT10, NIH NHP Reagent Resource, University of Massachusetts, Boston, MA), CCR7- BV711 (Clone G043H7, Biolegend). CD3-APCCy7 (Clone SP34-2, BD Biosciences), granzyme B-AF700 (Clone GB11, BD Biosciences), T-bet-PECy7 (Clone 4B10, eBioscience), Ki67-FITC (Clone B56, BD Bioscience) or Ki67-BV786 (Clone B56, BD Bioscience), CD69-APC (Clone FN50, BD Bioscience) or CD69-BV605 (Clone FN50, Biolegend), Perforin-FITC (Clone pf344, MabTech), TNF-BV605 (Clone MAb11, Biolegend), IFNg-BV785 (Clone 4S.B3, Biolegend), Live Dead Fixable Aqua Dead Cell Stain (Molecular Probes).

Roberts E.R., Carnathan D.G., Li H., Shaw G.M., Silvestri G, & Betts M.R. (2016). Collapse of Cytolytic Potential in SIV-Specific CD8+ T Cells Following Acute SIV Infection in Rhesus Macaques. PLoS Pathogens, 12(12), e1006135.

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Flow Cytometry Analysis of PBMCs

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PBMCs from a subset of patients undergoing THA or TKA were analyzed by flow cytometry (n = 19). Within 60 min after each draw, blood was layered over Ficoll-Paque PLUS, leukocytes were collected from the interface, and remaining red blood cells (RBCs) were lysed using RBC Lysis Buffer (BioLegend; San Diego, CA, USA). After lysis, cells were washed, incubated with Human FcR Binding Inhibitor (eBioscience; San Diego, CA, USA), and stained with anti-human CD8a-AlexaFluor488, CD4-PE, CD66b-PE-Dazzle, CD25-APC, CD45-APC-Cy7, CD127-PerCP-Cy5.5, CD14-PE-Cy7, HLA-DR-BV421, CD15-BV510, CD19-BV605, CD33-BV711, and CD16-BV650 (all from BioLegend; San Diego, CA, USA). Dead cells were excluded using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Life Technologies; Eugene, OR, USA) and analysis was performed using BD FACS-DIVA software, as previously described with the gating strategy depicted in Supplementary Figure S1 [16 (link)].

Heim C.E., Yamada K.J., Fallet R., Odvody J., Schwarz D.M., Lyden E.R., Anderson M.J., Alter R., Vidlak D., Hartman C.W., Konigsberg B.S., Cornett C.A., Garvin K.L., Mohamed N., Anderson A.S, & Kielian T. (2020). Orthopaedic Surgery Elicits a Systemic Anti-Inflammatory Signature. Journal of Clinical Medicine, 9(7), 2123.

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Multiparametric Analysis of SARS-CoV-2 Antibody Profiles

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Cryopreserved PBMCs were thawed and washed twice with 10 ml of fluorescence-activated cell sorting (FACS) buffer (1× PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 μl of PBS containing Zombie UV LIVE/DEAD dye at 1:200 dilution (BioLegend, #423108) and incubated at room temperature for 15 min. After washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD phycoerythrin (PE; Southern Biotech, #2030–09), IgM peridinin chlorophyll protein–Cy5.5 (BioLegend, #314512), CD20 allophycocyanin-H7 (BD Biosciences, #560734), CD27 PE-Cy7 (BioLegend, #302838), CD14 brilliant violet (BV) 650 (BioLegend, #301836), CD16 BV650 (BioLegend, #302042), IgG brilliant UV 395 (BD Biosciences, #564229), CD3 BV650 (BD Biosciences, #563916), CD21 PE-CF594 (BD Biosciences, #563474), Alexa Fluor 488–labeled Beta Spike protein (antibodies-online, #ABIN6963740), Alexa Fluor 647– labeled Omicron Spike protein (Sino Biological, #40589-V08H26), and BV421-l abeled Wuhan Spike protein (Sino Biological, #40589-V27B-B). All antibodies were used as per the manufacturer’s instruction, and the final concentration of each probe was 0.1 μg/ml. Cells were washed twice in FACS buffer and immediately acquired on a BD FACSAria III. FlowJo software v10 (TreeStar Inc.) was used for data analysis.

Arunachalam P.S., Feng Y., Ashraf U., Hu M., Walls A.C., Edara V.V., Zarnitsyna V.I., Aye P.P., Golden N., Miranda M.C., Green K.W., Threeton B.M., Maness N.J., Beddingfield B.J., Bohm R.P., Scheuermann S.E., Goff K., Dufour J., Russell-Lodrigue K., Kepl E., Fiala B., Wrenn S., Ravichandran R., Ellis D., Carter L., Rogers K., Shirreff L.M., Ferrell D.E., Deb Adhikary N.R., Fontenot J., Hammond H.L., Frieman M., Grifoni A., Sette A., O’Hagan D.T., Van Der Most R., Rappuoli R., Villinger F., Kleanthous H., Rappaport J., Suthar M.S., Veesler D., Wang T.T., King N.P, & Pulendran B. (2022). Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine. Science translational medicine, 14(658), eabq4130.

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