Macrophages were coincubated with PMA-stimulated neutrophils in the presence or absence of the inhibitors CytoB, DPI, or pyrochatechol for 4 hours, during the last 30 min of which, DNase I (10 U/ml) was added to remove extracellular NETs. The cells were then scraped and fixed for 15 min at room temperature with 2% PFA. The cells were permeabilized with 0.1% Triton X-100 for 5 min and subsequently blocked with 5% BSA for 20 min at room temperature. Cells were stained with anti-NE antibody (10 μg/ml) for 1 hour at room temperature. After three subsequent washes with PBS, the cells were stained with anti-rabbit Alexa Fluor 568 (2 μg/ml, Thermo Fisher Scientific) and CD66b-APCVio770 (Miltenyi) for 30 min at room temperature. After three washes with PBS, the cells were analyzed by flow cytometry.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.