Mouse placental and decidual macrophages were evaluated via flow cytometry as previously described with modification23 (link). Briefly, placenta and decidua were collected from each animal and pooled, weighed, minced with sterile scissors and digested in a solution containing 1 mg/mL collagenase, 1 mg/mL hyaluronidase, and 150 μg/mL DNAse I with agitation for 1 h at 37°C. Samples were washed using RPMI 1640 +/+ medium (containing 1% antibiotic and 10% fetal bovine serum), centrifuged at 1500 RPM at 4°C for 10 min followed by 100 μm nylon mesh filtration to eliminate remaining particulates. Cells were then washed and centrifuged on a Percoll gradient as previously described followed RBC lysis buffer (eBiosciences). After two more washes with RPMI 1640 +/+, cells were counted, and one million cells were aliquoted into flow cytometry tubes. Cells were surface stained for 20 min at 4°C with anti-mouse CD45 (Biolegend; Pacific Blue, clone 30-F11), anti-mouse CD11b (eBioscience; Alexa Fluor 700, clone M1/70), anti-mouse F4/80 (eBioscience; PE, clone BM8), and FcR blocking reagent (Miltenyi). Cells were then stained with a live/dead viability dye (Life Technologies; Aqua). Cells were resuspended in FACS buffer and immediately acquired on a BD LSR2 Flow Cytometer (BD Biosciences). Analysis was performed with BD FACS Diva software (BD Biosciences).