For the zymosan-induced peritonitis experiments, peritoneal exudate
cells harvested at the times indicated in the figure legend were washed in FACS
staining buffer (PBS containing 3% [vol/vol] 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 (PMNs). 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 vs. Y872F
MerTK-expressingMertk−/−macrophages, the cells were first incubated with 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 mean florescence intensity (MFI) of p-ERK and p-CaMKII in
APC-MerTK+ cells gated using a FACSCanto II (BD Biosciences) flow
cytometer and FlowJo software.
cells harvested at the times indicated in the figure legend were washed in FACS
staining buffer (PBS containing 3% [vol/vol] 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 (PMNs). 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 vs. Y872F
MerTK-expressingMertk−/−macrophages, the cells were first incubated with 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 mean florescence intensity (MFI) of p-ERK and p-CaMKII in
APC-MerTK+ cells gated using a FACSCanto II (BD Biosciences) flow
cytometer and FlowJo software.