Ly6g pacblue

Immediately following tissue processing described above, cell suspensions from the brain, galea, and bone flap were treated with TruStain F_cX (BioLegend Cat# 101320, RRID:AB_1574975) to prevent nonspecific antibody binding. A 5-color Ab panel was used to quantify leukocyte infiltrates that included CD45-APC (RRID:AB_312977), CD11b-FITC (RRID:AB_312789), Ly6G-PacBlue (BioLegend, Cat# 127612), Ly6C-PerCP-Cy5.5 (BD Biosciences, Cat# 560525), and F4/80-PECy7 (RRID:AB_893478). Nonviable cells were excluded from analysis using a Zombie UV Fixable Viability Kit (BioLegend, Cat# 423108), and samples were processed with a BD LSRII Green cytometer. Spherotech AccuCount Blank Particles (Cat# ACBP-100-10; 8.0-12.9 μm) were used to report absolute cell counts. Samples were analyzed with FlowJo (RRID:SCR_008520) using the gating strategy presented in Supplemental Fig. 1.

One day after i.p. injection of either CCL2 (50 μg) in PBS or PBS alone, mice were sacrificed, and peritoneal lavage fluid was analyzed by flow cytometry using the following commercial antibodies: anti-B220- FITC, F4/80-PE, Ly-6C-PerCP/Cy5.5, CD11b-PE/Cy7, Ly-6G-PacBlue, (Biolegend, San Diego, CA); and CCR2-allophycocyanin (APC; Bio-Techne (formerly R&D Systems), Minneapolis, MN). Cell samples were pre-incubated with 1 μg Fc-block (2.4G2 hybridoma; ATCC, Manassas, VA). Samples were acquired with a Becton Dickinson FACScan (BD Biosciences) flow cytometer with DxP multi-color upgrades by Cytek Development Inc. (Woodland Park, NJ), and data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR).