Cd66b pacific blue

Manufactured by BioLegend

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CD66b Pacific Blue is a fluorochrome-conjugated antibody that binds to the CD66b antigen, which is expressed on the surface of granulocytes. This product can be used for the identification and enumeration of granulocytes in flow cytometry analysis.

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Pacific Blue conjugated anti-CD66b (3 citations) Anti-human CD66b Pacific Blue (clone G10F5, (3 citations) Anti-CD66b-Pacific Blue (2 citations) Pacific blue CD66b (clone G10F5) (2 citations) Pacific Blue anti-CD66b (clone G10F5; (2 citations)

5 protocols using cd66b pacific blue

Immunophenotyping of Neutrophils and Monocytes

Cited 3 times

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The expression of TLR4, NOX2, and CD11b on the surface of neutrophils and monocytes was evaluated by flow cytometry. Whole blood (200 μL) was treated in 100-μL aliquots incubated at 37°C for 1 h untreated (vehicle) or with 10 ng/mL LPS (SIGMA Life Science, Ireland). Fluorochrome-conjugated monoclonal antibodies (mAb) specific for humans CD14-PerCP, CD15-PECy7, NOX2-FITC, CD66b-Pacific Blue, TLR4-APC (BioLegend®, USA), and CD11b-PE (BD Biosciences, UK) were used. The whole blood was then stained with mAb for 15 min. Red blood cells were lysed with BD lysis buffer. Cells were acquired on a FACSCanto II flow cytometer (BD Bioscience) and analyzed using FlowJo version 10 (Tree Star) (2 (link), 14 (link)). Neutrophils were delineated based on SSC-A and CD66b⁺ and monocytes based on SSC-A, CD66b⁻, and CD14⁺ (15 (link), 16 (link)). Monocytes were subdivided into classical, intermediate, and non-classical subtypes. Monocyte subsets were identified as classical: CD14^highCD16^neg/low; intermediate: CD14^highCD16^high; non-classical: CD14^lowCD16^high. A minimum of 10,000 events were collected, and relative expression of antigens was expressed as mean fluorescence intensity (MFI).

O'Dea M.I., Kelly L., McKenna E., Melo A.M., Ni Bhroin M., Hurley T., Byrne A.T., Colleran G., Vavasseur C., El-Khuffash A., Miletin J., Murphy J., Hickey F, & Molloy E.J. (2021). Dysregulated Monocyte and Neutrophil Functional Phenotype in Infants With Neonatal Encephalopathy Requiring Therapeutic Hypothermia. Frontiers in Pediatrics, 8, 598724.

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Antibody-Mediated Phagocytosis Assay

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Recombinant BDBV GP was biotinylated and conjugated to streptavidin-coated Alexa488 beads (Life Technologies). BDBV-coated beads were incubated with antibodies at 5 μg/ml in culture medium for 2 hours at 37°C. Human white blood cells were isolated from peripheral blood by lysis of red blood cells using ammonium chloride potassium lysis buffer. Cells were washed with PBS, and 5.0 x 10⁴ cells/well were added to bead-antibody immune complexes, and then incubated for 1 hour at 37°C. Cells were stained with the following antibodies to identify neutrophils: CD66b Pacific Blue (BioLegend clone G10F5), CD14 APC-Cy7 (BD Biosciences clone MφP9) and CD3 AlexaFluor700 (BD Biosciences clone UCHT1). Cells were fixed with 4% paraformaldehyde and were analyzed on a BD LSRII flow cytometer. A phagocytic score was determined as described above.

Ilinykh P.A., Santos R.I., Gunn B.M., Kuzmina N.A., Shen X., Huang K., Gilchuk P., Flyak A.I., Younan P., Alter G., Crowe JE J.r, & Bukreyev A. (2018). Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap. PLoS Pathogens, 14(8), e1007204.

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ADNP Assay for CHIKV Antibody Detection

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The ADNP assay was adapted from a previous publication (47 (link)). Briefly, recombinant, biotinylated CHIKV p62-E1 protein was conjugated to streptavidin-coated Alexa Fluor 488 beads (Invitrogen) for 2 h at 37°C. After washing two times in 0.1% BSA in PBS, CHIKV antigen–coated beads were incubated with hyperimmune IgG (5-fold dilutions) in cell culture medium for 2 h at 37°C to form immune complexes. Bone marrow cells were harvested from C57BL/6 mice (mADNP) or primary neutrophils were isolated from fresh ACD blood (hADNP). Cells were washed with PBS, and 5.0 × 10⁴ cells per well were added to the immune complexes and incubated for 1 h at 37°C. The following antibodies were used to stain the cells: mADNP: CD11c APC/Cy7 (clone N418; BioLegend), CD11b APC (clone M1/70; BioLegend), Ly-6C BV605 (clone HK1.4; BioLegend), Ly6G Pacific Blue (clone 1A8; BioLegend), and CD3 PE/Cy7 (clone 17A2; BioLegend) or hADNP: CD66b Pacific Blue (clone G10F5; BioLegend). Cells were fixed with 4% PFA and analyzed on a BD LSRII flow cytometer. Neutrophils were defined as CD3⁻ and CD11c⁻ cells that were Ly6C⁻, CD11b⁺, and Ly6G⁺ for mouse cells or CD66b⁺ for human cells. The phagocytic score was calculated as follows: (percentage of Alexa Fluor 488⁺ cells) × (Alexa Fluor 488 geometric mean fluorescent intensity of Alexa Fluor 488⁺ cells)/10,000.

Fox J.M., Roy V., Gunn B.M., Bolton G.R., Fremont D.H., Alter G., Diamond M.S, & Boesch A.W. (2023). Enhancing the therapeutic activity of hyperimmune IgG against chikungunya virus using FcγRIIIa affinity chromatography. Frontiers in Immunology, 14, 1153108.

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Antibody-Dependent Cellular Phagocytosis and Antibody-Dependent Neutrophil Phagocytosis Assays

Cited 1 time

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ADCP and ADNP were conducted as previously described [80 (link),81 (link)]. Briefly, spike or RBD proteins were biotinylated using EDC (Thermo Fisher Scientific) and EZ-link Sulfo-NHS-LC-LC (Thermo Fisher Scientific) and then coupled to yellow/green and then coupled to yellow/green FluoSphere NeutrAvidin-conjugated beads (Thermo Fisher Scientific, F8776). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADNP 1:50 dilution, ADCP 1:100 dilution) for 2 hours at 37°C and then washed to remove unbound antibody. The immune complexes were then incubated overnight with THP-1 cells (ADCP), or for 1 hour with RBC-lyzed whole blood (ADNP). THP-1 cells were then washed and fixed in 4% PFA, while the RBC-lyzed whole blood was washed, stained for CD66b Pacific Blue (BioLegend - San Diego, California, USA), CD3-AlexaFluro700 (BD Biosciences - Miami, Florida, USA), and CD14-APC-Cy7 (BD Biosciences) to identify neutrophils (CD3- CD14- CD66b+) and then fixed in 4% PFA. Flow cytometry was performed to identify the percentage of quantity of beads phagocytosed by THP-1 cells or neutrophils, and a phagocytosis score was calculated (% cells positive × median fluorescent intensity of positive cells). Flow cytometry was performed with an LSRII (BD), and analysis was performed using FlowJo V10.7.1.

Zhu D.Y., Gorman M.J., Yuan D., Yu J., Mercado N.B., McMahan K., Borducchi E.N., Lifton M., Liu J., Nampanya F., Patel S., Peter L., Tostanoski L.H., Pessaint L., Van Ry A., Finneyfrock B., Velasco J., Teow E., Brown R., Cook A., Andersen H., Lewis M.G., Lauffenburger D.A., Barouch D.H, & Alter G. (2022). Defining the determinants of protection against SARS-CoV-2 infection and viral control in a dose-down Ad26.CoV2.S vaccine study in nonhuman primates. PLoS Biology, 20(5), e3001609.

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Ebola Glycoprotein Phagocytosis Assay

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EBOV GP recombinant protein (IBT Bioservices) was biotinylated and
conjugated to streptavidin-coated Alexa488 beads (Life Technologies). EBOV
GP-coated beads were incubated with antibodies diluted in culture medium to
5μg/ml for 2 hours at 37°C. Human white blood cells were
isolated from peripheral blood by lysis of red blood cells using ammonium
chloride potassium (ACK) lysis buffer. Cells were washed with PBS, and 5.0
× 10⁴ cells/well were added to bead-antibody immune
complexes, and incubated for 1 hour at 37°C. Cells were stained with
the following antibodies to identify neutrophils: CD66b Pacific Blue
(BioLegend clone G10F5), CD14 APC-Cy7 (BD Biosciences clone MφP9),
and CD3 AlexaFluor700 (BD Biosciences clone UCHT1). Cells were fixed with 4%
paraformaldehyde and were analyzed on a BD LSRII flow cytometer. Data was
analyzed using FlowJo software, and a phagocytic score was determined using
the following calculation: (% of AlexaFluor488⁺cells)*(AlexaFluor488 geometric mean fluorescent intensity (MFI) of
AlexaFluor488⁺ cells)/10,000.

Bornholdt Z.A., Herbert A.S., Mire C.E., He S., Cross R.W., Wec A.Z., Abelson D.M., Geisbert J.B., James R.M., Rahim M.N., Zhu W., Borisevich V., Banadyga L., Gunn B.M., Agans K.N., Wirchnianski A.S., Goodwin E., Tierney K., Shestowsky W.S., Bohorov O., Bohorova N., Velasco J., Ailor E., Kim D., Pauly M.H., Whaley K.J., Alter G., Walker L.M., Chandran K., Zeitlin L., Qiu X., Geisbert T.W, & Dye J.M. (2019). A two-antibody pan-ebolavirus cocktail confers broad therapeutic protection in ferrets and nonhuman primates. Cell host & microbe, 25(1), 49-58.e5.

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