MPs were purified from treated cell supernatants as described above. After suspension of MPs in PBS, the MP numbers were quantitated by flow cytometry as described above. For experiments to digest DNA, MPs were treated with recombinant DNase I (rDNase; 10U rDNase / 4×106 MPs; Life Technologies) for 60 min at 37°C. After this treatment, the MPs were transferred to flow cytometry tubes at an MP density of 2×105/tube. To assess HMGB1 content, samples were stained with a Phycoerythrin (PE)-conjugated mouse anti-human HMGB1 antibody (2ul/tube; monoclonal antibody directed to residues 2–215, R&D systems, Minneapolis, MN) or a PE-conjugated mouse IgG2b (R&D Systems) for 60min at 20°C. To assess DNA, MPs were stained with a mouse monoclonal anti-dsDNA antibody (clone 163p.132; 4ug/tube; a kind gift from Dr. Tony Marion) or a mouse IgG2b (isotype control; 4ug/tube; Sigma-Aldrich). After this step, MPs treated with either anti-DNA or isotype control were incubated with a F(ab')2 sheep anti-mouse IgG PE (Sigma-Aldrich) for 30min at 20°C in the dark. The samples were then analyzed by flow cytometry.
Pe conjugated mouse igg2b
Manufactured by R&D Systems
Sourced in United States
PE-conjugated mouse IgG2b is a laboratory reagent used for flow cytometry applications. It consists of a mouse immunoglobulin G2b (IgG2b) antibody conjugated to the fluorescent dye phycoerythrin (PE).
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2 protocols using pe conjugated mouse igg2b
1
Quantification and Characterization of Extracellular Vesicles
2
TIM-1 Expression Analysis in Cells
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Sample cells were collected with the same density and washed with a staining buffer (PBS with 1% FBS). After centrifugation at 112× g for 5 min, the cells were re-suspended in a 100 μL staining buffer and stained with a 10 μL PE-conjugated anti-human TIM-1 antibody (R&D systems, clone#219211, Minneapolis, MN, USA) at 4 °C for 30 min. The negative controls were the unstained group (without antibody) and the isotype group, in which the cells were stained with the PE-conjugated mouse IgG2B (R&D systems, clone#133303, Minneapolis, MN, USA). Cells were washed with a 200 μL staining buffer twice, re-suspended in a 500 μL staining buffer, and temporarily stored at 4 °C. Cells were analyzed using a flow cytometer (Beckman coulter, CytoFLEX flow cytometer, Brea, CA, USA).
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