For CD63 measurements, PBCMCs, defined as CD117+ and CD203c+ cells, were dissolved in pre-warmed (37°C) Tyrode’s buffer at a concentration of 5x105 cells/mL. Next, 100 μL of the cells were stimulated with 100 µL tyrode buffer, anti-FcϵRI, substance P, succinylcholine, atracurium, ciprofloxacine or levofloxacin, for 3 and 20 min at 37°C. Reactions were stopped by placing the cells on ice. Subsequently, supernatants were removed by centrifugation (500 x g, 4°C, 5 min). Cells were stained with anti-human CD117-APC (clone 104D2, BD Biosciences), anti-human CD203c-PECy7 (clone NP4D6, eBioscience), anti-human MRGPRX2-PE (clone K125H4, BioLegend) and anti-human CD63-FITC (clone H5C6, BD Biosciences) for 20 min at 4°C. Next, cells were fixed with 1 mL Phosflow Lyse/Fix buffer (BD Biosciences) for 20 min. Finally, cells were washed and resuspended in PBS with 0.1% sodium azide and measured using flow cytometry.
Anti human cd63 fitc clone h5c6
Manufactured by BD
Sourced in United States
Anti-human CD63-FITC (clone H5C6) is a fluorescently labeled antibody that binds to the CD63 antigen on human cells. CD63 is a membrane glycoprotein that is commonly used as a marker for exosomes and other extracellular vesicles.
Automatically generated - may contain errors
Lab products found in correlation
Anti human mrgprx2 pe clone k125h4, by BioLegend (3 mentions)
Anti human cd203c pecy7 clone np4d6, by Thermo Fisher Scientific (3 mentions)
Anti human cd117 apc clone 104d2, by BD (2 mentions)
Phosflow lyse fix buffer, by BD (2 mentions)
Anti human fcεri pe clone aer 37, by Thermo Fisher Scientific (2 mentions)
Anti human cd300a pe clone e59, by Beckman Coulter (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti human cd25 fitc clone b1.49.9, by Beckman Coulter (1 mentions)
Alexa fluor 405, by Thermo Fisher Scientific (1 mentions)
Anti ige, by BD (1 mentions)
Anti human ige clone ge 1, by Merck Group (1 mentions)
5 protocols using anti human cd63 fitc clone h5c6
1
Flow Cytometric Analysis of CD63 Expression
2
Platelet Activation by GPVI Agonist
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Diluted platelet-rich plasma (1:10 with Tyrode’s buffer) was treated with increasing concentrations of the collagen receptor GPVI selective agonist convulxin (DSM, Nutritional Products Ltd., Branch Pentapharm, 4147 Aesch BL, Switzerland) for 6 min at room temperature and fixed with 0.5% (v/v) formaldehyde (final concentration) for 30 min at room temperature. Platelets were washed once with Tyrode’s buffer and subsequently centrifuged at 800× g for 10 min at room temperature. Pelleted platelets were incubated with anti-human CD62P-FITC (clone AK-4, 5 µg/mL, BD Biosciences) and anti-human CD63-FITC (clone H5C6, 5 µg/mL, BD Biosciences) antibodies for 30 min at room temperature. Platelets were analyzed by flow cytometry as described in Section 2.11 . Data were presented as linear values of the mean fluorescence intensity (MFI).
3
Immunophenotyping of Cultured Mast Cells
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Cultured cells were stained for surface makers with monoclonal anti-human CD117-APC (clone 104D2, BD Biosciences, Erembodegem, Belgium), anti-human CD203c-PECy7 (clone NP4D6, eBioscience, San Diego, USA), anti-human FcεRI-PE (clone AER-37, Thermofisher Scientific, Waltham, USA), anti-human MRGPRX2-PE (clone K125H4, BioLegend, San Diego, USA), anti-human CD300a-PE (clone E59.126, Beckman Coulter, California, USA), anti-human CD32-PE (FLI8.26, BD Biosciences) and anti-human CD63-FITC (clone H5C6, BD Biosciences). Cells were stained for 20 min at 4°C in the dark. For staining of intracellular tryptase and chymase, cells were fixed with 1 mL of 4% paraformaldehyde solution for 30 min at room temperature, washed with and resuspended in PBS with 0.05% Triton-X-100 (pH=7.4) (Sigma-Aldrich). Next, cells were stained with monoclonal anti-human tryptase-Alexa Fluor 700 (antitryptase-AF700) (BioLegend) and monoclonal anti-human chymase-Alexa Fluor 700 (anti-chymase-AF700) (BioLegend), respectively, and incubated at 37°C for 30 min in the dark. Cells were washed and resolved in PBS with 0.1% sodium azide and analyzed. Representative plots of the immunphenotyping are shown in figure 1 of the online repository.
4
Multiparameter Characterization of Cultured Cells
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Cultured cells were stained for surface makers with monoclonal anti-human CD117-APC (clone 104D2, BD Biosciences, Erembodegem, Belgium), anti-human CD203c-PECy7 (clone NP4D6, eBioscience, San Diego, USA), anti-human FcεRI-PE (clone AER-37, Thermofischer Scientific), anti-human MRGPRX2-PE (clone K125H4, BioLegend, San Diego, USA), anti-human CD300a-PE (clone E59.126, Beckman Coulter, California, USA), anti-human CD32-PE (clone FLI8.26, BD Biosciences), anti-human CD63-FITC (clone H5C6, BD Biosciences) and anti-human CD25-FITC (clone B1.49.9, Beckman Coulter). Cells were stained for 20 minutes at 4°C in the dark, washed, and resuspended in PBS supplemented with 0.1% sodium azide (PBS_NaN3).
Additional staining for the basophilic markers CD123 and CD11b was performed to evaluate the potential presence of basophil-like cells 14, 34, 35 . In preliminary experiments, intracellular staining of trytase and chymase was performed, as previously described 27, 30 .
Additional staining for the basophilic markers CD123 and CD11b was performed to evaluate the potential presence of basophil-like cells 14, 34, 35 . In preliminary experiments, intracellular staining of trytase and chymase was performed, as previously described 27, 30 .
5
Basophil Activation Assay with Moxifloxacin
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Basophil activation experiments were performed as described in (15, 16) . Briefly, aliquots of heparinized whole blood where incubated with 12.5, 125, 625 or 1.250 µmol/L of moxifloxacin (Avelox® 400mg/250 mL, Bayer SA-NV). Dilution buffer and anti-IgE (10 g/mL, clone G7-18; mouse IgG2a; Pharmingen, BD Biosciences, Erembodegem, Belgium) served as a negative and positive control, respectively. Reactions were stopped by chilling on ice, adding ice-cooled PBS-EDTA 10 mmol/L EDTA and spinning. To select and quantify basophil activation, cells were stained with anti-human IgE (clone GE-1, Sigma Aldrich GmBH, Steinheim, Germany) labeled with Alexa-Fluor 405 (Molecular Probes, Invitrogen, Paisley, UK) and anti-human CD63-FITC (clone H5C6, BD Bioscience), CD203c-APC (clone NP4D6, BD Bioscience) on ice. Cells were lysed/fixed with Phosflow Lyse/Fix buffer (BD Biosciences) and subsequently were washed twice with PBS with 0.1% Triton-X-100 (PBS-TX, pH=7.4). To stain intracellular histamine, V500-labeled DAO (BD Biosciences) was added and incubated at 37°C (45 min). Cells were washed and re-suspended with PBS with sodium azide and measured.
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