Computational protocol: N-Acetylcysteine Amide Protects Against Oxidative Stress–Induced Microparticle Release From Human Retinal Pigment Epithelial Cells

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Protocol publication

[…] Isolated MPs were stained with the following antibody-fluorophores in varying combinations with compensation and IgG controls used where necessary: annexin V-FITC, annexin V-PE, PI, CD46-APC, CD55-PE, CD59-APC, Milk fat globule-epidermal growth factor (EGF) factor 8 (MFG-E8)-FITC, and duramycin-FITC (). Controls for IgG1 and IgG2a conjugated to APC were used. In some cases, MPs were exposed to 16 μM, 100-fold excess compared with MFG-E8, cRGD for 30 minutes prior to staining with MFG-E8-FITC. Annexin V and PI staining was performed at room temperature for 15 minutes per the manufacturer's instructions while all other staining was performed on ice for 1 hour. Samples were run on a LSR II flow cytometer (BD Biosciences, San Jose, CA, USA; Becton Dickinson) equipped with 450, 488, and 633 nm lasers with a side-scatter threshold set to 750. Acquisition was performed with BD FACSDiva software. The injection port was wiped and water was run through the cytometer between samples to minimize cross-contamination of samples. FlowJo version 10 (FlowJo, LLC, Ashland, OR, USA) was used to analyze and quantify data. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Homo sapiens
Diseases Glycogen Storage Disease Type II, Retinal Diseases
Chemicals Hydrogen Peroxide