5x10E4 to 1x10E5 cells were labelled for 30 minutes in 18 µl to 100 µl PBS + 1% BSA containing one or combinations of the following antibodies: anti-CD16-PECy7 (Biolegend; 1:1000; 3G8), anti-CD62 L-APCCy7 (Biolegend; 1:25; Greg-56), anti-CD63-APC (Biolegend; 1:100; H5 C6), anti-CD66b-PE (Biolegend; 1:100; G10F5), DAPI (Sigma-Aldrich), anti-CD3-PacificBlue (Beckman Coulter, 1: 50, UCHT-1), anti-CD14-FITC (Miltenyi, 1:100, Tük 4), anti-ICAM-1-AlexaFluor-405 (SantaCruz, 1:100, sc-107-af405), an in-house VHH against P-selectin (1:125, B10.6 [43 (link)],), or Annexin-V (250 ug/ml, VPS diagnostics, cat. nr. A705). Neutrophil activation was assessed by measuring the increase in CD62L expression [44 (link)] and the simultaneous decrease in CD16 and increase in CD63 expression [45 (link),46 (link)]; HUVEC activation was assessed by evaluating the increase in ICAM-1 expression [47 (link)]; red blood cell activation was assessed by evaluating phosphatidylserine exposure detected using annexin-V staining [48 (link)]; platelet activation was assessed by investigating P-selectin expression [49 (link)]. Surface labelling of cells was measured on a FACSCanto (BD Biosciences) or a LSR-II (BD Biosciences) flow cytometer relative to unlabelled controls.
Anti cd63 apc
Manufactured by BioLegend
Sourced in United States
Anti-CD63-APC is a fluorescently-labeled antibody that specifically binds to the CD63 protein. CD63 is a membrane glycoprotein that is involved in vesicle trafficking and is commonly used as a marker for exosomes and lysosome-related organelles.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto, by BD (2 mentions)
Anti cd14 fitc, by Miltenyi Biotec (1 mentions)
Anti cd62l apc cy7, by BioLegend (1 mentions)
Anti cd16 pecy7, by BioLegend (1 mentions)
Anti cd66b pe, by BioLegend (1 mentions)
Anti cd3 pacificblue, by Beckman Coulter (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti cd81 apc, by BioLegend (1 mentions)
0.2 μm filter, by Merck Group (1 mentions)
Facsdiva software, by BD (1 mentions)
Facs fortessa, by BD (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Percpcy5.5 anti cd11b, by BioLegend (1 mentions)
Apc anti cd66b, by BioLegend (1 mentions)
6 protocols using anti cd63 apc
1
Multiparameter Flow Cytometry Analysis of Cellular Activation
2
Flow Cytometric Analysis of Extracellular Vesicles
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The flow cytometry method was applied to determine the expression of CD63, CD9, and CD81 on the surface of isolated particles. Isolated TEx or TMv were resuspended in PBS filtered through 0.2 μm filters (Merck Millipore) and then labeled with monoclonal antibodies conjugated with fluorochromes: anti-CD63 APC, anti-CD9 APC, anti-CD81 APC, rat IgG2aκ APC and Armenian hamster IgG APC isotype controls (all from BioLegend). The expression of cell surface markers was analyzed using FACS Fortessa with FACSDiva software (Becton Dickinson).
3
Neutrophil CD63 Expression Assay
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Neutrophils were pre-incubated with 20 ng/mL TNFα (1029-TA-050, R&D Systems, Minneapolis, MN, USA) for 30 min and then 10 μM cytochalasin B (CB, HY-16928, MCE, Shanghai, China) for 5 min at 37 °C and stimulated with 6-OAU of various concentrations for 15 min at 37 °C. Then the reaction was stopped with ice-cold PBS buffer and cells were incubated at 4 °C for 30 min with anti-CD63 APC (143906, Biolegend, San Diego, CA, USA). The level of CD63 on the cell surface was analyzed by flow cytometry.
4
Peanut-sensitized Murine Mast Cell Activation
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Murine bone marrow-derived mast cells were cultured from wild-type BALB/c mice, as described in Stassen et al. 28 Cells were sensitized against peanut by incubation with serum derived from peanut-sensitized mice (ratio 1:10 in medium) overnight. After washing, mast cells were challenged with peanut extract (in a concentration of 1 mg/mL) preincubated with serum of naive mice or with serum of CuMVtt-Ara h 1-vaccinated mice (ratio 1:10 in medium) for 30 minutes in the incubator (378C). After washing, cells were stained with anti-CD63-APC (BioLegend) to detect activation. Measurements were performed with FACS Canto (BD Biosciences) and analysis with FlowJo software (FlowJo LCC).
5
Phenotyping of Activated Neutrophils
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1x10^6 PMNs were incubated with the following surface antibodies for 20 min on ice in the dark. Surface antibodies included PerCP-Cy5.5-anti-CD11b, APC-anti-CD63, APC-anti-CD66b (all Biolegend). To exclude dead cells, a fixable viability dye was included. Flow cytometry was performed using an LSR II (BD Bioscience) and analyzed with FlowJo (TreeStar).
6
Immunophenotyping of Small Extracellular Vesicles
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For flow cytometry analysis, pellets obtained from 10 ml of medium were resuspended in 100 μl of PBS. sEVs were stained with APC anti-CD63 (4 μg/ml; BioLegend, 143905) for 30 min at 4°C in darkness. After incubation, positive events were read by FACSVerse flow cytometry. One unstained sample of sEVs and one sample with the antibody at 4 μg/ml in PBS were used as negative controls. Calibration of the cytometer was assessed using fluorescent particles of standardized size (Nano Fluorescent Particle Size Standard Kit, Spherotech).
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