Kaluza for gallios software version 1

Isolated CD11b⁺ cells from brain or BM were fixed and permeabilized using fixation/permeabilization solution (BD Biosciences, San Jose, CA, USA) for 20 min at 4°C. After washing, the cells were incubated with anti-Iba1 antibody (Wako) for 30 min at 4°C followed by incubation with Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific) for 30 min at 4°C. The median of fluorescent intensity (MFI) was measured in total 5,000-count cells using a Beckman Coulter Gallios flow cytometer and Kaluza for Gallios software version 1.3 (Beckman Coulter, Indianapolis, IN, USA).

Six hours after the last LPS treatment, expressions of cell surface antigens CD86 and Mrc1 were analyzed by flow cytometry as previously described^{5 (link)} with minor modifications. In brief, cells were detached by 0.25% trypsin treatment (Life Technologies) and resuspended in PBS containing 0.5% bovine serum albumin and 2 mM EDTA. The cells were stained with FITC-labeled anti-CD86 antibody (eBioscience, San Diego, CA, USA) or APC-labeled anti-Mrc1 antibody (BioLegend) for 30 min at 4 °C. The MFI was measured in a total of 50,000-counted cells using a Beckman Coulter Gallios flow cytometer and Kaluza for Gallios software version 1.3 (Beckman Coulter, Indianapolis, IN, USA).