Basophil activation tests (BAT) were performed using the Flow CAST Basophil Activation Test kit (Bühlmann Laboratories, Schönenbuch, Switzerland). 50 μl aliquots of whole blood from peanut allergic patients were pre incubated with 125-1000 μg/ml of purified IgA for 2-4 hours at 37°C in 100 μl of basophil stimulation buffer. Samples were incubated for l5 min at 37°C with 3.6x10-4 μg/ml of crude peanut extract (CPE) and staining antibodies for human PE anti-CCR3 (Biolegend, San Diego, CA) and FITC anti-CD63 (BD Biosciences, Franklin Lakes, NJ). Anti-FcϵRI stimulation was used as a positive control for basophil activation. After red blood cell lysis, cells were washed and assessed for activation by flow cytometry. Basophils were identified as SSClowCCR3+ and activated cells identified based on CD63 expression. Around 200 basophils were evaluated for each sample. Peripheral blood from peanut allergic donors was obtained with informed consent under a protocol approved by the Institutional Review Board of Boston Children’s Hospital.
Fitc anti cd63
Manufactured by BD
Sourced in United States
FITC anti-CD63 is a fluorescently labeled antibody that binds to the CD63 protein. CD63 is a transmembrane glycoprotein commonly used as a marker for exosomes and other extracellular vesicles.
Automatically generated - may contain errors
Lab products found in correlation
Protein g sepharose column, by GE Healthcare (1 mentions)
Pe anti fcεri, by BioLegend (1 mentions)
Sorvall mtx 150, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Annexin 5 apoptosis detection kit efluor 450, by Thermo Fisher Scientific (1 mentions)
Anti cd81 apc, by BD (1 mentions)
4 protocols using fitc anti cd63
1
Basophil Activation Test for Peanut Allergy
2
Monoclonal Antibody Characterization for Fel d 1 Allergy
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The monoclonal Fel d 1-specific IgE and IgG antibodies used in these experiments have been described previously (14) . They have been genetically engineered to be expressed as either human or mouse antibodies and were produced in CHO cells (Evitria AG, Zürich, Switzerland) and purified by affinity chromatography over a protein L or protein G Sepharose column (GE Healthcare) respectively. The monoclonal Fel d 1-specific IgE F127 was chosen for subsequent activation and binding assays in this study. For activation assays, the mast cells were stained with FITC anti-CD63 (BD Bioscience, NJ, USA) antibodies. Marker expressions were assessed with APC anti-c-kit (Biolegend, San Diego, CA, USA), PE anti-FcεRI (Biolegend) and IgE binding with APC anti-IgE (Miltenyi Biotec, Bergisch Gladbach, Germany). Mast cell binding of Fel d 1 was assessed by staining with the FITC-labelled monoclonal mouse anti-Fel d 1 IgG recognizing the non-overlapping epitope G078 (14) .
3
Exosome Isolation and Characterization
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Exosomes were isolated using a multi-step process. Large vesicles were eliminated by sequential centrifugation up to 50,000g as described12 (link). The remaining particles were pelleted by ultracentrifugation (Sorvall mTx 150, Thermo Scientific, Springfield, NJ) at 100,000g for 18 h. Vesicles expressing CD63 were immunoselected using CD63 magnetic bead isolation. The recovered particle size was verified by nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Amesbury, UK) as described11 (link). The data were analyzed with the NTA software (NANOSight version 2.3) using dilutions with deionized water. Three videos at a minimum of 200 completed tracks were collected at 30-s time intervals/video per sample.
Exosomes were further distinguished by flow cytometry with magnetic beads coupled to anti-CD9-PE, anti-CD63-FITC and anti-ALIX-APC (BD Biosciences).
Exosomes were further distinguished by flow cytometry with magnetic beads coupled to anti-CD9-PE, anti-CD63-FITC and anti-ALIX-APC (BD Biosciences).
4
Multiparametric Flow Cytometry Analysis
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Single‐cell suspensions were analysed by a BD FACS Canto II flow cytometer equipped with 488, 633, and 405 nm lasers using FACS Diva software (Becton Dickinson, Franklin Lakes, NJ, USA) as described before (Berger et al, 2014 ). Propidium iodide (PI) and apoptosis stainings were performed as described previously (Berger et al, 2014 ) and according to manufacturer's instructions (Annexin V Apoptosis Detection Kit eFluor® 450, 88‐8006; eBioscience, San Diego, CA, USA). eGFP+ or mCherry+ KU812 cells were high‐purity FACS sorted on a FACS Aria III equipped with a 488 nm laser at 4°C (Becton Dickinson). Exosomes were stained with anti‐CD63‐FITC (561924; BD Biosciences) and anti‐CD81‐APC (561958; BD Biosciences).
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