Cd16 bv605

Manufactured by BioLegend

CD16 BV605 is a fluorescent-conjugated antibody that recognizes the CD16 receptor expressed on the surface of certain immune cells, such as natural killer cells and subsets of monocytes and neutrophils. It is used in flow cytometry applications for the identification and analysis of these cell populations.

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4 protocols using cd16 bv605

Phenotypic Analysis of Cryopreserved PBMCs

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Cryopreserved PBMCs were thawed and washed twice with 10 mL of FACS buffer (1 x PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 uL of 1x PBS containing Zombie UV live/dead dye at 1:200 dilution (BioLegend, 423108) and incubate at room temperature for 15 minutes. Following washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD PE (BD Biosciences, 555779), IgM PerCP-Cy5.5 (BioLegend, 314512), CD20 APC-H7 (BD Biosciences, 560734), CD27 PE-Cy7 (BioLegend, 302838), CD14 PE/Dazzle^™ 594 (BioLegend, 301852), CD16 BV605 (BioLegend, 302040), IgG BV650 (BD Biosciences, 740596), CD3 BUV737 (BD Biosciences, 612750) and Alexa Fluor 488-labeled Wuhan spike (SinoBiological, 40589-V27B-B), and BV421 labeled Omicron Spike (SinoBiological^™, 40589-V49H3-B). All antibodies were used as the manufacturer’s instruction and the final concentration of each probe was 0.1 ug/ml. Cells were washed twice in FACS buffer and immediately acquired on a BD FACS Aria III for acquisition and FlowJo for analysis.

Wimmers F., Burrell A.R., Feng Y., Zheng H., Arunachalam P.S., Hu M., Spranger S., Nyhoff L., Joshi D., Trisal M., Awasthi M., Bellusci L., Ashraf U., Kowli S., Konvinse K.C., Yang E., Blanco M., Pellegrini K., Tharp G., Hagan T., Chinthrajah R.S., Grifoni A., Sette A., Nadeau K.C., Haslam D.B., Bosinger S.E., Wrammert J., Maecker H.T., Utz P.J., Wang T.T., Khurana S., Khatri P., Staat M.A, & Pulendran B. (2023). Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life. medRxiv.

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Characterization of NK Cell Activation

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Cryopreserved immune cells were thawed and enriched for NK cells using the EasySep™ Human NK Cell Enrichment Kit (STEMCELL Technologies, Cambridge, MA). Enriched NK cells were cultured with or without K562 cells at a 1:1 concentration for 4 hours with Golgi Stop added after 1 hour. Cells were then prepared for flow cytometry with the following markers: Live/Dead Blue (Invitrogen, Waltham, MA), CD56 APC, CD18 APC-Cy7, CD16 BV605 (Biolegend, San Diego, CA), CD3 BUV395, CD7 BV711, CD107 PE (BD, Franklin Lakes, NJ), and IFITM1 FITC (Biorbyt, St. Louis, MO).

Hilliard K.A., Throm A.A., Pingel J.T., Saucier N., Zaher H.S, & French A.R. (2022). Expansion of a novel population of NK cells with low ribosome expression in juvenile dermatomyositis. Frontiers in Immunology, 13, 1007022.

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Multiparametric Flow Cytometry of Isolated Cells

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Single cell isolations from biopsies were analyzed by flow cytometry immediately after isolation. Biopsies and blood or isolated neutrophils from the bacteria stimulations were stained using the following surface antigen mouse anti-human antibodies with clone denoted in (), from Becton Dickinson, and Co. (BD) Biosciences (Franklin, NJ) unless otherwise stated: CD45 PE-CF594 (HI30),CD11b APC-Cy7 (ICRF44), CD66b PE (Biolegend, G10F5), CD49d PE/Cy5 (Biolegend, 9F10), CD20 Brilliant Violet 570 (Biolegend, 2H7), CD3 Brilliant Violet 570 (Biolegend, UCHT1), CD16 BV605 (3G8), CD15 BV650 (HI98), and CD14 BV786 (M5E2). Following surface staining, cells were permeabilized using Cytofix/Cytoperm (BD Biosciences). Intracellular active Caspase-3 was stained using a v450-conjugated rabbit anti-human antibody (BD Biosciences, C92-605). Stained samples were fixed in 1% paraformaldehyde and collected on an LSR II (BD Biosciences, La Jolla, California). Analysis was performed in FlowJo (version 9.7.6, Treestar Inc., Ashland, Oregon). Samples with less than 100 events in the neutrophil gate were not included in analyses due to an inability to ensure adequate fluorescence separation of populations and therefore accurate gating of the neutrophil cell population.

Hensley-McBain T., Wu M.C., Manuzak J.A., Cheu R.K., Gustin A., Driscoll C.B., Zevin A.S., Miller C.J., Coronado E., Smith E., Chang J., Gale M J.r., Somsouk M., Burgener A.D., Hunt P.W., Hope T.J., Collier A.C, & Klatt N.R. (2019). Increased mucosal neutrophil survival is associated with altered microbiota in HIV infection. PLoS Pathogens, 15(4), e1007672.

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Phagocytic Assay with E. coli Bioparticles

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Blood was collected into 3.8% sodium citrate for use with pHrodo red Escherichia coli bioparticles (ThermoFisher). Whole blood was incubated at 37°C with R406 or vehicle (0.01% DMSO) for 15 minutes followed by incubation with bioparticles for 30 minutes. Samples were incubated on ice as a negative control. Samples were then used for cell surface staining with CD14 (BV421), CD16 (BV605), and CD66b (APC; BioLegend) followed by red blood cell lysis. Samples were analyzed using a BD LSR Fortessa flow cytometer.

Prendecki M., Gulati K., Pisacano N., Pinheiro D., Bhatt T., Mawhin M., Toulza F., Masuda E.S., Cowburn A., Lodge K.M., Tam F.W., Roufosse C., Pusey C.D, & McAdoo S.P. (2022). Syk Activation in Circulating and Tissue Innate Immune Cells in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis. Arthritis & Rheumatology (Hoboken, N.j.), 75(1), 84-97.

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