Ly6g bv510

Bone marrow cells were flushed with ice-cold HBSS^–++, filtered through 40 μm cell strainers, counted, pelleted at 326 × g for 10 min at 4°C, and resuspended in ice-cold DPBS⁺⁺⁺⁺ at 4 × 10⁷ cells/ml. 125 μl of cells were either kept on ice, or were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF, or with 1 μg/ml LPS, for 45 min at 37°C. Cells were sedimented at 10,000 × g for 30 s at 4°C, resuspended in Fc block (BD Biosciences, clone 2.4G2, 1:1000; for Mac1 and L-selectin) or in DPBS⁺⁺⁺⁺ (for FcγRIII), and incubated on ice for 15 min. Cells were sedimented at 10,000 × g for 30 s, resuspended in ice-cold DPBS⁺⁺⁺⁺ containing fixable viability dye (eBioscience, eFluor™ 780, 1:1000), antibodies for neutrophil markers Ly6G (Ly6G-BV510, BioLegend, clone 1A8, 1:500) and Mac1 (CD11b-AF647, BD Bioscience Clone M1/70, 1:1000), and PE-labelled antibodies for FcγRIII (CD16, BioLegend, clone S17014E, 1:100) or L-selectin (BD Biosciences clone MEL-14, 1:100), and were incubated on ice for 30 min. Cells were washed in ice-cold HBSS^–++, 1 mM EDTA, resuspended in 300 μl ice-cold HBSS^–++, 1 mM EDTA, and kept on ice. Flow cytometry was performed using a BioRad ZE5 flow cytometer, recording 20,000 neutrophils per sample. Neutrophils were identified by Ly6G^hi, CD11b^hi staining, and the mean fluorescence intensity (mfi) of receptor levels on the neutrophil surface was quantitated using FlowJo.

Cells were stained using the following fluorophore conjugated anti-mouse antibodies: WGA-488 (Invitrogen), Ly6G-FitC (clone 1A8, BioLegend), Ly6G-BV510 (clone 1A8, BioLegend), Ly6G-PE (clone 1A8, BioLegend), CD11c-BV605 (clone N418, BioLegend), F4/80-FitC (clone BM8, BioLegend), F4/80-PE (clone BM8, BioLegend), CD11b-PE (clone M1/70, BioLegend), Ly6C-PE Cy7 (clone HK1.4, BioLegend), CD63-APC (clone NVG-2, BioLegend), CD9-APC (clone MZ3, BioLegend), IL-1α-PE (clone ALF-161, BioLegend), and IL-1β-APC (clone NJTEN3, ThermoFisher Scientific). Antibodies were diluted in wash buffer (PBS with 1% BSA and 2 mM EDTA). Cells were stained for 20 minutes at 4°C, washed with wash buffer, fixed for 15 minutes in BD Biosciences cytofix/cytoperm, and permeabilization prior to intracellular staining. ACEA Novocyte was used for flow cytometry, and Novoexpress software was used for subsequent analysis. AMNIS ImageStream was used for imaging flow cytometry and the AMNIS IDEAS software was used to calculate the colocalization coefficient.

Tumor cell suspensions (4 × 10⁶) prepared by mechanical and enzymatic dissociation^{16 (link)} were stained in 96-wells round bottom plates with live/dead staining (Blue fluorescent reactive dye, #L34962 Invitrogen) during 20 min at room temperature. For flow-cytometry analysis and sorting, Fc receptors were blocked with anti-FcR (anti-CD16 and CD32, at 5 µg/ml, Biolegend #101339). After two washes in PBS 2% FCS, cells were stained with the following antibodies (all used at 1:100): anti-CD11b-BV421 (#562605), CD64-APC (#558539), CD11c-PeCy7 (#557401), TCRβ-BV605 (#562840) and CD4-BV711 (#563050) all purchased from BD Pharmingen; anti-CD45-AF700 (#103128), Ly6C-APCCy7 (#128025), Ly6G-BV510 (#127633), F4/80 BV650 (#123149), CD206-PE (#141706), IA/IE-BV785 (#107645) all purchased from Biolegend, and CD8-PerCPef710 (#46-0081-82) purchased from eBioscience. After washing, cells were fixed in 1% PFA, stored at 4 °C, and acquired the next day on LSR II or FORTESSA (BD Bioscience). For detection of phosphorylated proteins, cell suspensions were stimulated 3 h with DMXAA 250 µg/ml, fixed immediately in PFA 4%, permeabilized with frozen methanol 90%, stained overnight with 1:100 pTBK1-PE (#13498) antibodies (purchased from Cell signaling), then washed and further stained for multicolor flow cytometry.

For flow cytometric analyses, C57BL/6 mice were infected with 200 TgVEG WT-GFP-LUC or TgVEGΔROCY1-GFP-LUC parasites in 200 µL of PBS via intraperitoneal injection. At day 28 post-infection, mice were transcardially perfused with 50 mL of 1X PBS to remove non-adherent blood cells. Brains were harvested and digested using Dispase II (Roche Applied Science) diluted in Hepes-buffered saline. To remove myelin, 35% and 75% percoll (GE Healthcare) gradients were used. To block non-specific binding of antibodies to immune cells, the isolated cells were resuspended in 10% TrueStain FcX Buffer (BioLegend) in staining buffer (3% fetal bovine serum in 1X PBS). Cells were surface-stained with fluorescent dye-conjugated antibodies diluted in staining buffer (Biolegend: Ly6G:BV510 Cat#127633, CD11b:BV605 Cat#101257, CD45:BV785 Cat#103149, Ly6C:PerCp-Cy5.5 Cat#128017, CD3:APC-Cy7 Cat#100222). Cells were then resuspended in 1X PBS and run on the Novocyte flow cytometer (Agilent). Flow cytometry data were analyzed and graphically represented utilizing FlowJo software (Treestar), and the gating strategy is outlined in Fig. S6.

Twenty-four hours after stroke, mice were euthanized, and single-cell suspensions were made of the ischemic (ipsilesional) hemispheres as previously described (68 (link)). Cells were incubated with CD45 APC-Cy7 (103116, BioLegend), CD11b PE-Cy7 (25-0112-82, eBioscience), Ly6G BV510 (127633, BioLegend), and CD16/CD32 (14-0161-82, Fc block, eBioscience) for 30 minutes at room temperature in PBS plus 5% FBS. Thirty minutes later, cells were washed, fixed, and run on a Beckman Coulter Cytoflex located in the Utah Flow Cytometry Core.

Twenty-four hours after CLP or sham surgery, total white blood cell and differential neutrophil counts were determined both in whole blood samples collected from orbital vein using heparinized capillaries tubes and peritoneal fluid by washing with 3 mL of PBS, respectively. Then, samples were incubated with CD45 APC-Cy7 (Biolegend), CD11b PE-Cy7 (EBioscience), Ly6G BV510 (Biolegend), and CD16/CD32 (Fc-block, EBioscience) for 30 minutes at 37^°C. Thirty minutes later, cells were washed, fixed, and ran on a Beckman Coulter Cytoflex located in the University of Utah Flow Cytometry Core.

Corneas from infected mice were dissected and incubated with 3 mg/ml collagenase (C0130; Sigma-Aldrich) in RPMI (Life Technologies), with 1% HEPES (Life Technologies), 1% penicillin-streptomycin (Life Technologies), and 0.5% BSA (Fisher Bioreagents) for 1 h and 15 min at 37◦C. All subsequent steps were at 4⁰C. Cells were recovered following centrifugation and incubated 5 min with anti-mouse CD16/32 Ab (BioLegend) to block Fc receptors, then 20 min with anti-mouse CD45-allophycocyanin, Ly6GBV510, Ly6C-PE-Cy7, CD11b-PETxRed, CCR2-BV421, or F4/80-FITC (BioLegend) and fixable viability dye (BD Biosciences). Cells were washed with FACS buffer and quantified using an ACEA Novocyte flow cytometer and NovoExpress software. The gating strategy identified total cells in infected corneas by forward and side scatter, followed by gating on single cells and live cells were identified using the E780 viability dye (Biolegend). Neutrophils were identified as CD45 + , CD11b + , Ly6G + , CCR2-, and monocytes were CD45 + , CD11b + , Ly6G- CCR2 + . The gating strategy is shown in Fig. S3K.