Fortessa facs machine

Liver samples were minced and pressed through a 70-μm cell strainer. Cell suspensions were washed once in RPMI-1640 medium and centrifuged at 2000 rpm, 4 °C for 5 min. The pellets were suspended with 40% Percoll solution in RPMI-1640 medium and overlaid on 70% Percoll solution in RPMI-1640 medium and centrifuged at 400 × g (up 6, down 0), 4 °C for 20 min. Hepatic mononuclear cells were obtained from the interphase. Single-cell suspensions were washed with FACS washing buffer (2% FBS, 0.1% NaN₃ in PBS) once and incubated with fluorescence-conjugated antibodies against cell surface molecules for 30 min on ice in the presence of 2.4G2 monoclonal antibody to block FcγR binding. Isotype antibodies were included as negative controls. After washing with FACS buffer, cells were fixed with 1% (w/v) paraformaldehyde in PBS and preserved at 4 ºC. Flow cytometry was performed using a Becton Dickinson FACS Fortessa machine (East Rutherford, NJ, USA) and data were analyzed using the FlowJo version 10. CD45-APC (30-F11, 103112), CD11b-PE-Cy7 (M1/70, 101216), Gr1-PE (RB6-8C5, 108408), F4/80-FITC (BM8, 123108), Ly6C-BV605 (HK1.4, 128036), Ly6G-BV421 (1A8, 127628), DR5-PE (MD5-1, 119906), and CD3-PERCP (17A2, 100218) were purchased from BioLegend (San Diego, CA, USA).

Flow cytometry was used to determine macrophage and microglia populations in single cell preparations of the hippocampus prepared in complete media; IMDM (GIBCO/Invitrogen; Carlsbad, CA), 10% FCS, and P/S on ice. Samples were stained with an antibody mix (MACS buffer +1% inactivated Rat serum, 1% α-FcγII/III (clone 2.4G2), α-F4/80 (clone GK1.5; BD Pharmingen^TM, FITC), and α-Iba1 (clone GR106078-1; Abcam®, FITC) for 30 min on ice, and then fixed in 2% paraformaldehyde before permeabilization (0.5 g saponin, 0.055 g CaCl2, 0.0625 g MgSO4, 0.25 g NaN3, 0.5 g BSA, 10 mM HEPES in final volume of 500 ml of 1X PBS) for 1 hour at 4 °C. Intracellular staining of samples was done with an antibody mix (MACS buffer +2% inactivated Rat serum, 2% α-FcγII/III (clone 2.4G2), α-IL-1α (clone ALF-161; BioLegend®, PE); α-IL-6 (Lot 99523; BP Pharmingen^TM, PE); α-iNOS2 (clone Sc651; Santa Cruz Biotechnology®), α-MHCII (clone M5/114.15.2; eBioscience®, AlexaFluor 700), α-IL-4 (clone 11B11; eBioscience®, APC); α-IL-13 (clone eBio13A; eBioscience®, PE Cy7); and α-Arginase1 (clone H1010; Santa Cruz Biotechnology®) for 30 min on ice and read by a Becton Dickinson FACS FORTESSA machine (BD San Diego, CA). Data was analyzed by FlowJo© Treestar (Ashland, OR) and graphed with GraphPad Prism® software. Unless otherwise stated, antibodies were from BD Pharmingen^TM.

Single-cell suspensions were washed once with FACS washing buffer (2% FBS and 0.1% NaN₃ in PBS). Cells were then incubated with fluorescence-conjugated antibodies against cell surface molecules for 30 min on ice. PERCP-CY5.5 antimouse CD45 (Cat# 103131, clone 30-F11), PE antimouse Ter119 (Cat# 116207, clone TER-119), Brilliant Violet 510 antimouse CD11b (Cat#101263, clone M1/70), Brilliant Violet 421 antimouse CD11c (Cat#117330, clone N418), APC-Cyanine7 antimouse Ly6G (Cat# 108424, clone RB6-8C5), and Brilliant Violet 605 antimouse Ly6C (Cat# 108440, clone HK1.4) were purchased from BioLegend. To determine cell viability, a LIVE/DEAD^™ Fixable Green Dead Cell Stain Kit (Cat# L34970, Thermo Fisher Scientific, Waltham, MA, United States) was used according to the manufacturer’s instructions. After washing with FACS buffer, the cells were fixed with 1% (w/v) paraformaldehyde in PBS and preserved at 4 °C. Flow cytometry was performed using a Becton Dickinson FACS Fortessa machine (East Rutherford, NJ, United States) and the data were analyzed using the FlowJo version 10.