Ghost dyetm violet 510

Macrophages were mechanically harvested after 7 to 12 days of differentiation/treatment using a cell scraper (Greiner Bio-one, Kremsmünster, AT), spun at 1000 rpm/10 min, and analyzed for cell integrity and viability by microscopy using Trypan blue exclusion or a Live/Dead cell viability dye (Ghost Dye^TM Violet 510, Tonbo Biosciences, San Diego, CA, USA). For immunophenotyping, viable cells were stained using specific fluorophore-conjugated anti-human monoclonal antibodies (Table 1) at concentrations recommended by the manufacturer. Cells were incubated with Abs for 30 min at +4 °C in the dark, washed using PBS/0.1% BSA (FACS Wash) buffer, and fixed in 4% Paraformaldehyde (Biolegend, San Diego, CA, USA) before analyzing at BD FACS Verse^TM flow-cytometer (BD Biosciences, San Diego, CA, USA). Live/dead dye was routinely applied to exclude dead cells before analyzing cells of interest. Data were analyzed using FACS Suite or Diva software v 9.0 (BD Biosciences, San Diego, CA, USA).

Antibiotics including vancomycin (V2002-5G), neomycin (N6386-100G), and pimaricin (P9703-25MG) were purchased from MilliporeSigma (Burlington, MA, USA). FITC-dextran was purchased from MilliporeSigma (Burlington, MA, USA), and ELISA kits for TNF-α, IL-1β, and IL-10 were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against GFAP and Aβ were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). The anti-Iba1 antibody was purchased from Wako Chemicals USA, Inc. (Richmond, VA, USA). Elite Vectastain ABC reagents, Vector VIP, biotinylated anti-rabbit, and anti-mouse antibodies were purchased from Vector Laboratories Inc (Burlingame, CA, USA). ZymoBIOMICS DNA Kits D4300 (Zymo, Irvine, CA, USA), A Quantibody^® Mouse Cytokine Array (QAM-TH-17; RayBiotech, Inc., Norcross, GA, USA), and blood and chocolate agar plates (Thermo Fisher Scientific, Rockford, IL, USA) were also used. Ghost dye^TM Violet 510 was purchased from Tonbo Biosciences (San Diego, CA, USA). The antibodies and the clones used in flow cytometry experiments are detailed in Table 1.