Antibody solutions were centrifuged at 14,000 × g for 1 h at 4 °C to remove aggregates before use. EVs (1–2 μg EV proteins, as measured on Nanodrop, in 20 μL of PBS) were blocked using 1 μg of mouse serum IgG for 10 min at RT then incubated with: AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G, 0.4 µg/2 µg EVs), APC mouse antihuman CD81 (Clone JS-81 (RUO)) (BD Biosciences, 561958, 1 µL/2 µg EVs), AF-647 mouse antihuman CD63 (Clone H5C6 (RUO)) (BD, Biosciences, 561983, 2 µL/2 µg EVs), AF-488 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003G, 0.4 µg/2 µg EVs), APC isotype control mouse IgG1κ (Clone MOPC-21 (RUO)) (BD Biosciences, 555751, 1 µL/2 µg EVs) or AF-647 isotype control mouse IgG1κ (Clone MOPC-21 (RUO)) (BD, Biosciences, 557714, 2 µL/2 µg EVs) for 45 min at 4 °C. The solution was then diluted to 200 μL with PBS and the samples were run on Apogee A50 microflow cytometer (MFC) (Apogee Flow Systems, Hertfordshire, UK) (http://www.apogeeflow.com/products.php ). The reference ApogeeMix beads (Apogee Flow Systems, 1493), were used to assess the performance of Apogee MFC and to compare the size distribution of the EVs. PBS was run as a background control. Data were analyzed using Flow Jo v10.6.2.
Af 488 isotype control mouse igg2b
Manufactured by R&D Systems
AF-488 isotype control mouse IgG2b is a laboratory reagent used as a control in immunoassays and flow cytometry experiments. It is a mouse immunoglobulin G2b (IgG2b) antibody conjugated to the Alexa Fluor 488 fluorescent dye. This reagent can be used to help determine the level of non-specific binding in experiments involving mouse IgG2b antibodies.
Automatically generated - may contain errors
2 protocols using af 488 isotype control mouse igg2b
1
Characterization of Extracellular Vesicles by Flow Cytometry
2
Exosome Characterization by Flow Cytometry
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Antibody solutions were centrifuged at 14000 × g for 1 h at 4 °C to remove aggregates before use. Exosomes (1-2 μg protein equivalent amount of exosomes in 20 μL of PBS) were blocked using 1μg of mouse serum IgG for 10 min at RT then incubated with: AF-488 mouse anti-human ACE2 (R&D systems, FAB9333G), APC mouse antihuman CD81 (BD Biosciences, 561958), AF-647 mouse antihuman CD63 (BD, Biosciences, 561983), AF-488 isotype control mouse IgG2b (R&D systems, IC003G), APC isotype control mouse IgG1κ (BD Biosciences, 555751) or AF-647 isotype control mouse IgG1κ (BD, Biosciences, 557714) for 45 min at 4°C. The solution was then diluted to 200μL with PBS and the samples were run on Apogee A50 Micro Flow Cytometer (MFC) (Apogee Flow Systems, Hertfordshire, UK) (http://www.apogeeflow.com/products.php). The reference ApogeeMix beads (Apogee Flow Systems, 1493), were used to assess the performance of Apogee MFC, and to compare the size distribution of the exosomes. PBS was run as a background control.
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