For the zymosan-induced peritonitis experiments, peritoneal exudate cells harvested at the times indicated in the figure legend were washed with fluorescence-activated cell sorting (FACS) staining buffer [PBS containing 3% (v/v) FBS]. The cells were incubated for 5 min at 4°C with Fc Block (BD Biosciences) and then labeled with Pacific Blue–Ly6G (clone 1A8, eBioscience) to detect polymorphonuclear neutrophils. To detect p-CaMKII in exudate macrophages, cells were stained with PE-F4/80 (clone BM8, eBioscience) for 30 min at 4°C to stain macrophages, which was followed by fixation and permeabilization of the cells. Permeabilized cells were then incubated with rabbit anti–p-CaMKII for 1 hour at 4°C and then with Alexa Fluor 647–conjugated goat anti-rabbit secondary antibody for 30 min at 4°C. For the flow cytometric assay of p-ERK and p-CaMKII in WT MerTK–expressing versus Y872F MerTK–expressing Mertk−/− macrophages, the cells were first incubated with allophycocyanin (APC)–MerTK (clone #125518, R&D Systems) to label transduced macrophages. After fixation and permeabilization, the cells were incubated with rabbit anti–p-ERK or anti–p-CaMKII antibodies, which was followed by incubation with PE-conjugated anti-rabbit secondary antibody. The cells were suspended in FACS buffer and analyzed for the MFI of p-ERK and p-CaMKII in APC-MerTK+ cells gated using a FACSCanto II (BD Biosciences) flow cytometer and FlowJo software.

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.