Cd11c alexafluor700

Manufactured by BioLegend

CD11c AlexaFluor700 is a fluorescently-labeled antibody that targets the CD11c cell surface antigen. It can be used for the identification and analysis of cells expressing CD11c, such as dendritic cells and certain types of macrophages, using flow cytometry or other fluorescence-based techniques.

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5 protocols using cd11c alexafluor700

Antibody-mediated phagocytosis of CHIKV

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Recombinant CHIKV p62-E1 was biotinylated and conjugated to streptavidin-coated Alexa488 beads. CHIKV p62-E1-coated beads were incubated with five-fold dilutions of antibodies (mAbs: 1 μg/ml to 0.0016 μg/ml) in culture medium for 2 h at 37°C. Bone marrow cells were harvested from C57BL/6 mice. 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: CD11b APC (BioLegend clone M1/70), CD11c AlexaFluor700 (BioLegend clone N418), Ly6G BV421 (BioLegend clone 1A8). Cells were fixed with 4% PFA and were analyzed on a BD LSRII flow cytometer. A phagocytic score was determined as described above.

Fox J.M., Roy V., Gunn B.M., Huang L., Fong R.H., Edeling M.A., Mack M., Fremont D.H., Doranz B.J., Johnson S., Alter G, & Diamond M.S. (2019). Optimal therapeutic activity of monoclonal antibodies against chikungunya virus requires Fc-FcγR interaction on monocytes. Science immunology, 4(32), eaav5062.

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Comprehensive Immune Cell Profiling

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All cells were stained with Live/Dead Fixable Aqua (1:2000; Invitrogen) at room temperature for 30 min in the dark. Following the Live/Dead viability stain, cells were incubated with anti-mouse CD16/CD32 (Fc block; 1:100, eBioscience), CD3 PE-eFluor610 (1:100; eBioscience), CD4 PE (1:100; eBioscience), CD8b APC-eFluor780 (1:100; eBioscience), CD11b PE-Cy7 (1:200; eBioscience), Ly6G Pacific Blue (1:100; BioLegend), CD45 PerCP-Cy5.5 (1:100; eBioscience), CD11c Alexa Fluor 700 (1:50; BioLegend) Ly6C FITC (1:200; eBioscience) in FACS buffer. Samples were run on an LSRII (BD Biosciences) flow cytometer and analyzed with FlowJo_V10 software.
Single cell lymphocytes were gated on forward scatter area (FSA; size) and side scatter area (SSA; granularity) and then by forward scatter height (FSH) and FSA. Live cells were selected as the negative Fixable Aqua population. CD45⁺ cells were selected and gated by CD3 (T cells) versus CD11b (myeloid cells). The CD3⁺ population was then gated on CD4 (helper T cells) and CD8 (cytotoxic T cells). CD11b⁺ cells were further gated on CD11c and Ly6G for the following populations: CD11c⁻Ly6G⁻ (microglia and macrophages), CD11c-Ly6G⁺ (neutrophils), and CD11c⁺Ly6G⁻ (dendritic cells). CD11c⁻Ly6G⁻ cells were gated for CD45 and low and high populations were further gated for MHCII and Ly6C to determine their activation state (Suppl. Fig. S1).

Eidson L.N., Gao Q., Qu H., Kikuchi D.S., Campos A.C., Faidley E.A., Sun Y.Y., Kuan C.Y., Pagano R.L., Lassègue B., Tansey M.G., Griendling K.K, & Hernandes M.S. (2021). Poldip2 controls leukocyte infiltration into the ischemic brain by regulating focal adhesion kinase-mediated VCAM-1 induction. Scientific Reports, 11, 5533.

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Quantifying Fc-Mediated Phagocytosis of CHIKV

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Recombinant CHIKV p62-E1 was biotinylated and conjugated to streptavidin-coated Alexa488 beads (Life Technologies). CHIKV p62-E1-coated beads were incubated with five-fold dilutions of antibodies (mAbs: 1 μg/ml to 0.0016 μg/ml) in culture medium for 2 h at 37°C. Monocytes harvested from the bone marrow of C57BL/6 mice and purified using a CD14+ enrichment kit (Stem Cell Technologies) were added at a concentration of 2.5 × 10⁴ cells/well and incubated for 4 h at 37°C in low adherence 96-well plates. Following incubation, monocytes were incubated with the following antibodies: CD11b APC (BioLegend clone M1/70), CD11c AlexaFluor700 (BioLegend clone N418), and Ly6G BV421 (BioLegend clone 1A8). Cells were fixed with 4% PFA and analyzed by flow cytometry on a BD LSRII using Diva and FlowJo analysis software. The phagocytic score was determined using the following calculation: (% of AlexaFluor488⁺ cells) x (AlexaFluor488 geometric mean fluorescent intensity (MFI) of AlexaFluor488⁺ cells)/10,000.

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Antibody-Dependent Cellular Phagocytosis Assay

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The ADCP assay was adapted from a previously described method (45 (link)). Briefly, recombinant biotinylated CHIKV p62-E1 conjugated to streptavidin-coated Alexa Flour 488 beads (Invitrogen) for 2 h at 37°C and washed two times in 0.1% BSA in PBS. CHIKV p62-E1-coated beads were incubated with polyclonal antibodies (5-fold dilutions) in culture medium for 2 h at 37°C. The human THP-1 monocytic cell line (hADCP) or monocytes harvested and purified from the bone marrow of C57BL/6 mice using a CD14⁺ enrichment kit (Stem Cell Technologies) (mADCP) were added at a concentration of 2.5 × 10⁴ cells/well and incubated for 4 h at 37°C (mADCP) or overnight (hADCP) in low adherence 96-well plates (mADCP) or regular 96-well assay plates (hADCP). Following incubation, mouse monocytes (mADCP) were incubated with the following antibodies: Ly6G BV421 (BioLegend clone 1A8), CD11c AlexaFluor700 (BioLegend clone N418), and CD11b APC (BioLegend clone M1/70). After fixing the cells with 4% PFA, they were analyzed by flow cytometry (BD LSRII). The data was collected using Diva and analyzed using FlowJo software. The phagocytic score was calculated as follows: (% of Alexa Fluor 488 cells) x (Alexa Fluor 488 geometric mean fluorescent intensity (MFI) 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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Splenocyte Activation Assay

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Splenocytes (5 × 10⁶ cells) from mice at 0 (uninfected controls) and 8 days post infection were harvested 2 h after challenge with LPS and stained with fluorescein-labeled monoclonal antibodies (mAbs) specific for cell surface markers or cytokines. The following flow cytometry mAbs specific were used: CD11c-Alexa fluor 700 (1:100), MHC II-APCCy7 (1:200), F4/80- APC (Biolegend, 1:200), CD11b-PECy7 (1:4000), pro-IL-1β-FITC (eBioscience, 1:100), and AmCyan (Live/Dead kit, ThermoFisher). The intracellular fixation and permeabilization buffer set of eBioscience (ThermoFisher) were used to perform pro-IL-1β stain.

Pereira L.M., Assis P.A., de Araújo N.M., Durso D.F., Junqueira C., Ataíde M.A., Pereira D.B., Lien E., Fitzgerald K.A., Zamboni D.S., Golenbock D.T, & Gazzinelli R.T. (2020). Caspase-8 mediates inflammation and disease in rodent malaria. Nature Communications, 11, 4596.

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