Cd16 pe

Manufactured by BioLegend

Sourced in United States

CD16-PE is a fluorochrome-conjugated monoclonal antibody that binds to the human CD16 antigen, also known as FcγRIII. CD16 is a low-affinity Fc receptor that is expressed on natural killer cells, monocytes, and a subset of T cells. The PE fluorescent label allows for the detection and enumeration of CD16-positive cells using flow cytometry.

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Lab products found in correlation

Cd56 pe, by BioLegend (2 mentions) Cd56 apc, by BioLegend (2 mentions) Cd14 fitc, by BioLegend (2 mentions) Cytofix cytoperm kit, by BD (2 mentions) Attune nxt, by Thermo Fisher Scientific (2 mentions) Cd36 pe, by BioLegend (1 mentions) Cd15 apc, by BioLegend (1 mentions) Hla dr apc, by BD (1 mentions) Facscalibur, by BD (1 mentions) Igg1 apc, by BD (1 mentions) Ifn γ fitc, by BD (1 mentions) Igg1 fitc, by BD (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Cd56 apc, by Thermo Fisher Scientific (1 mentions) Hla dr pe, by BioLegend (1 mentions) Il 6 pe, by Thermo Fisher Scientific (1 mentions) Rat igg1 pe, by Thermo Fisher Scientific (1 mentions) Cd4 apc, by BioLegend (1 mentions) Golgistop, by BD (1 mentions) Golgiplug, by BD (1 mentions)

Close spelling variants of this product

CD16-PE-Cy7 (14 citations) Anti-CD16 PE (5 citations) CD16-PE-Cy5 (5 citations) PE/Cyanine7 anti-human CD16 antibody (3 citations) PE anti-human CD16 Antibody (3 citations) Anti-CD16-PE-Cy5 (2 citations) Mouse anti-human PE-Cy7 CD16(Clone 3G8) (2 citations) Anti-human CD16-PE (2 citations) PE-conjugated anti-CD16 antibody (2 citations) Anti-CD16-PE-Cy7 (clone 3G8, (2 citations)

16 protocols using cd16 pe

Identifying Immune Cell Subsets by Flow Cytometry

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Cells were washed to remove DTT and stained with the indicated antibodies: HLA-DR-APC (BD Bioscience, Oxford, UK), CD16-PE, CD36-PE, CD15-APC (Biolegend, San Diego, CA) and CD14-biotin (Southern Biotech, Birmingham, AL). Staining combinations were as follows: CD15, CD14/CD16, CD14/HLA-DR/CD36. Streptavidin-PE-Cy5.5 tandem conjugate (eBioscience) was used as a secondary reagent to detect biotinylated primary antibodies. Following staining, samples were immediately analysed by flow cytometry (FACScalibur, Becton Dickinson, Oxford, UK).
Neutrophils were identified as CD15⁺ events. Cells of the monocyte/macrophage lineage were identified as CD14⁺. CD15⁺ neutrophils were CD14 negative, allowing distinction of these two cell types [10] (link). CD14⁺/16⁺⁺ non-classical monocytes were identified as highly CD16 positive CD14⁺ cells. Forward scatter (FSC) and side scatter (SSC) profiles were also examined. Flow cytometry data were analysed using FlowJo (Tree Star Inc, Ashland, OR).

Prince L.R., Maxwell N.C., Gill S.K., Dockrell D.H., Sabroe I., McGreal E.P., Kotecha S, & Whyte M.K. (2014). Macrophage Phenotype Is Associated with Disease Severity in Preterm Infants with Chronic Lung Disease. PLoS ONE, 9(8), e103059.

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CFSE Proliferation Assay with Cytokine Stimulation

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CFSE staining was done using 5 µM CFSE (Invitrogen, Cat# C34554) as described by Parish et al. [42] (link) and stimulated with cytokines for 6 days prior to flow cytometric analysis. Antibodies used: CD8-APC (BD, Cat# 345775), CD3-APC (BD, Cat# 555335), CD4-APC (Biolegend, Cat# 300514), CD56-APC (eBioscience, Cat#17-0569-42), CD16-PE (Biolegend, Cat#302007), HLA-DR-PE (Biolegend, Cat# 307605), NKG2D-APC (RnD Systems, Cat# FAB139A). For intracellular staining, IL-6-PE (eBioscience, Cat#12-7069) and IFN-γ-FITC (BD, Cat# 554700) were used. Isotype controls were IgG1 APC (BD, cat # 555751), rat IgG1-PE (Invitrogen, Cat # R104) and IgG1-FITC (BD, Cat #555748). PBMCs were incubated for 6 h +/− LPS (Sigma-Aldrich, L2654, 1 µg/mL) and cells were stained using the BD Cytofix/Cytoperm Kit (BD, Cat# 554714), Golgistop (BD, Cat# 554724) or Golgiplug (BD, Cat# 555029) according to the manufacturer’s protocol. Samples were run on an Accuri C6 Flow cytometer and data were analyzed using FCS Express v. 3.

Reichwald K., Jørgensen T.Z., Tougaard P, & Skov S. (2014). TL1A Induces TCR Independent IL-6 and TNF-α Production and Growth of PLZF+ Leukocytes. PLoS ONE, 9(1), e85793.

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Immunophenotyping of Bone Marrow Cells

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Immunophenotyping of bone marrow (BM) was performed using an FC500 or Facscalibur flow cytometer. FACS buffer was made with PBS+2mM EGTA+2% FBS. Primary AML and leukemic stem cell fractions were detected using the following antibodies (company; product #; clone): anti-CD34 PE (BD bio-sciences; 348057; 8G12), anti-CD34 FITC (BD Pharmingen; 555821; 581), anti-CD38 APC (ebiosciences; 17-0389-42; HIT2) and anti-CD123 PE (BD Pharmingen; 558714; 7G3), anti-CD45 APC (BD Pharmingen; 557513; TU116) and anti-class I HLA A, B, C (Biolegend; 311404; W6/32). NK-92 cells lines were assessed for CD16 expression using CD16 PE (Biolegend; 302008; 3G8). Leukemia cell lines were evaluated using anti-CD123 PErCy5.5 (BD Biosciences; 560904; 7G3). Cell sorting was performed using a FacsAria cell sorter as described in the Online Supplementary Methods.

Williams B.A., Wang X.H., Leyton J.V., Maghera S., Deif B., Reilly R.M., Minden M.D, & Keating A. (2018). CD16+NK-92 and anti-CD123 monoclonal antibody prolongs survival in primary human acute myeloid leukemia xenografted mice. Haematologica, 103(10), 1720-1729.

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Characterization of Human Leukocyte Subsets

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Human leukocyte suspensions were plated in round bottom 96-well plates (Corning)
at ≤10⁶ cells/25 μL flow buffer and blocked using 10
μg/mL human TruStain FcX blocking solution (BioLegend, San Diego, CA).
Cells were stained for 30 minutes in the dark at 4°C in a 50 μL final
volume using a cocktail of fluorescently labeled antibodies against cell surface
markers suspended in flow buffer (1X PBS, 2% FBS, and 1% sodium azide). After
staining, cells were washed with 200 μL flow buffer, pelleted, resuspended
in 185 μL of BD stabilizing fixative (BD Biosciences), and transferred for
analysis to polystyrene tubes (12 × 75 mm) (Becton Dickinson, Franklin
Lakes, NJ). Cells were analyzed on a BD FACSCanto or LSRFortessa flow cytometer
with FACSDiva software (BD Biosciences) and analyzed by FlowJo (FlowJo, Ashland,
OR). Fluorophore-conjugated antibodies were CD45-BV421 (BioLegend, catalogue
#304032), CD16-PE (BioLegend, catalogue # 302056), CD11b-PerCP-Cy/5.5
(BioLegend, catalogue #301328), control IgG-PE-Cy7 (BioLegend, catalogue
# 400125), and CD15-PE-Cy7 (BD, catalogue # 560827). Peripheral
neutrophil levels were gated as previously described^{7 (link)}; CNS neutrophils were gated using CD45, CD11b,
and CD15. A nonspecific IgG control was used for CD15 staining to subtract
nonspecific events.

Murdock B.J., Goutman S.A., Boss J., Kim S, & Feldman E.L. (2021). Amyotrophic Lateral Sclerosis Survival Associates With Neutrophils in a Sex-specific Manner. Neurology® Neuroimmunology & Neuroinflammation, 8(2), e953.

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Multiparametric Flow Cytometry Analysis

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To determine the size of peripheral blood leukocyte and BM nucleated cell populations, cells were labeled with the following antibodies against human cell surface markers: anti-human cluster of differentiation (CD)235a-phycoerythrin (PE), CD14-PE-cyanin 5, CD16-PE, CD20-PE, CD203c-PE, CD3-PE-cyanin 5, CD4-PE-cyanin 5, CD4-PE-cyanin 7, CD8-PE-cyanin 5, and CD56-PE (all from BioLegend, San Diego, CA, USA); CD71-PE-cyanin 5, CD3-PE, and CD11c-PE (all from BD Biosciences); and CD141-allophycocyanin (Miltenyi Biotec, Bergisch Gladbach, Germany). The total lymphocyte population was determined from forward and side scatter, and CD4+ and CD8+ T cells, natural killer (NK) cells, and B cells were separated into CD3+/CD4+, CD3+/CD8+, CD56+/CD3−, and CD20+ populations, respectively. The monocyte population was determined from forward and side scatter and by CD14 expression. The granulocyte population was gated by forward and side scatter, and neutrophils and eosinophils were separated according to CD16 expression (CD16+ and CD16−, respectively). Basophils were isolated as the CD203c+ population. Plasmacytoid DCs, CD1c+ myeloid DCs, and CD141^high myeloid DCs were isolated from peripheral blood mononuclear cells on a FACSAriaII cell sorter as previously described [39 (link)]. Erythroid cells present in the BM cell population were gated according to CD235 and CD71 (TFR1) expression.

Sakamoto S., Kawabata H., Masuda T., Uchiyama T., Mizumoto C., Ohmori K., Koeffler H.P., Kadowaki N, & Takaori-Kondo A. (2015). H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner. PLoS ONE, 10(10), e0139915.

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Whole Blood Immune Cell Phenotyping

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Whole blood (100 μL) was stained for 20 minutes with either CD14 FITC and CD16 PE or antihuman CD14-PerCP and CD206-Alexafluor 488 with appropriate isotype controls [Biolegend, San Diego, USA, 13]. Cells were then washed twice with PBS, resuspended in 400 μL of PBS and acquired in a Flow Cytometer.

Mukhopadhyay D., Mukherjee S., Roy S., Dalton J.E., Kundu S., Sarkar A., Das N.K., Kaye P.M, & Chatterjee M. (2015). M2 Polarization of Monocytes-Macrophages Is a Hallmark of Indian Post Kala-Azar Dermal Leishmaniasis. PLoS Neglected Tropical Diseases, 9(10), e0004145.

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Multiparametric Flow Cytometry of Immune Cells

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Cells were stained with Ghost-Dye/V510, CD3 + /V421, CD4 + /FITC, CD8 + /APC-Cy7, CD16 + /PE, CD19 + /PE-Cy7 and CD56 + /APC (Biolegend). Cells were fixed in Fixation and Permeabilization Solution (BD). A Cytoflex S flow cytometer (Beckman Coulter) was used to analyze the subpopulation ratios.

Morandini F., Rechsteiner C., Perez K., Praz V., Lopez Garcia G., Hinte L.C., von Meyenn F, & Ocampo A. (2023). ATAC-clock: An aging clock based on chromatin accessibility. GeroScience, 46(2), 1789-1806.

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Leukocyte Surface Marker Staining

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100 μl of DAF-2DA loaded blood was aliquoted into 12 × 75 mm tubes for leukocyte surface marker staining for monocytes (CD14-AF647, Biolegend, CA, USA, M5E2 clone), granulocytes (CD16 PE, Biolegend, 3G8 clone), and natural killer cells (CD56 PECy3, Biolegend, MEM-188 clone) antibodies used per manufacturer’s instructions. Briefly, tubes with NO (DAF-2DA) and surface antibodies and controls were incubated for 30 min in the dark, and red blood cells were lysed for 8 min in the dark with 3 ml RBC lysis buffer (0.15 M Ammonium Cl, 10 mM Potassium Bicarbonate, 0.1 mM EDTA). Tubes were centrifuged at 300 × g for 10 min, supernatant was carefully removed, and cell pellet washed 2 × with 4 mL of wash solution (1XPBS-no Ca or Mg, 1% heated inactive fetal calf serum, 0.02% NaN3) and centrifuged again as above. Cells were resuspended in 500 μL of 1 × phosphate buffer saline (1XPBS Irvine Scientific, Irvine, CA, USA). Samples were read on the BD C6 Accuri flow cytometer as described in Section 2.6.

Maharaj S., Lu K.D., Radom-Aizik S., Zaldivar F., Haddad F., Shin H.W., Leu S.Y., Nussbaum E., Randhawa I, & Cooper D.M. (2017). Inter- and intra-subject variability of nitric oxide levels in leukocyte subpopulations. Nitric oxide : biology and chemistry, 72, 41-45.

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Fc Receptor Blocking and Multicolor Flow Cytometry

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Human TruStain FcX Fc Receptor Blocking Solution (BioLegend Cat#422302) was added according to the number of cells, mixed well and incubated for 10 min at room temperature. Cells were divided into separate microfuge tubes for staining with fluorescently labeled antibodies (CD3-FITC, CD45-PerCP, CD16-PE, and CD14-APC-Cy7 from BioLegend) for flow cytometry and incubated on ice for 30 min. 1 mL of FACS buffer (heat inactivated FBS, 0.5M EDTA pH8.0 1x PBS) was added to the cells, mixed well and centrifuged at 300 rcf for 5 min at 4°C. The supernatant was removed and the cells were washed once more. The final cell pellet was resuspended in 100 μL of FACS buffer and kept on ice in the dark. Sytox blue (0.2 μL/100 μL or less) was added directly before flow cytometry analysis and analyzed on a Thermo Fisher Attune NxT at the Cornell BRC Flow Cytometry Facility.

Vu L.T., Ahmed F., Zhu H., Iu D.S., Fogarty E.A., Kwak Y., Chen W., Franconi C.J., Munn P.R., Tate A.E., Levine S.M., Stevens J., Mao X., Shungu D.C., Moore G.E., Keller B.A., Hanson M.R., Grenier J.K, & Grimson A. (2024). Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation. Cell Reports Medicine, 5(1), 101373.

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Comprehensive Immune Cell Phenotyping

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To analyze cell surface activation markers, PBMCs were stimulated by 1 μM TLR7/8 agonists or DMSO (as a negative control) for 24 h at 37°C. Then cells were collected and washed in FACS buffer (PBS + 2%FBS). Cells were incubated by fluorescence antibodies at 4°C for 30 min. For T cells, CD3-PE-Cy7 (Biolegend), CD4-PE (BD), CD8-FITC (Biolegend), CD25-BV421 (Biolegend), CD69-BV510 (Biolegend). For NK cells, CD16-PE (Biolegend), CD56-APC (Biolegend), CD69-BV510 (Biolegend), NKG2D-BV421 (Biolegend). For monocytes, CD14-PE-Cy7 (Invitrogen), CD40-APC-Cy7 (Biolegend) HLR-DR-BV510 (Biolegend). For dendritic cells, anti-human lineage cocktail-APC (Biolegend), CD11c-PE-Cy7 (Biolegend), CD80-BV510 (Biolegend), CD86-AF488 (Biolegend).

Li Y., Wang Z., Hou Y., Liu X., Hong J., Shi X., Huang X., Zhang T., Liao X, & Zhang L. (2023). Novel TLR7/8 agonists promote activation of HIV-1 latent reservoirs and human T and NK cells. Frontiers in Microbiology, 14, 1033448.

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