Ly6c percp cy5

Liver nonparenchymal cells were incubated with Fc block and incubated in FACS buffer with fluorophore conjugated antibodies on ice for the following macrophage panel: F480 (A700, 1:200, Biolegend), CD64 (PE, 1:200, BD Biosciences), CD11b (FITC, 1:200, BD Biosciences), Ly6C (PerCP-Cy5.5, 1:200, BD Biosciences), CLEC4F (Unconjugated, 1:200, R&D Systems). Following antibody incubation samples were washed twice with FACS buffer. After washing, CLEC4F samples were incubated with a secondary antibody (Donkey anti-goat AF647, 1:500, ThermoFisher) and washed twice after incubation. The Becton Dickinson LSRII machine at the NCSU Flow Cytometry Core was used to acquire all flow data. Flow data was analyzed using FlowJo software v10.8.

RPMI 1640, FBS, penicillin, and streptomycin were obtained from Invitrogen (Grand Island, NY). The liver dissociation kit, recombinant murine was from Miltenyi Biotec. Phorbol 12-myristate 13-acetate (PMA), brefeldin A, ionomycin, CD3, CD28, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). We obtained the following fluorescein-conjugated anti-mouse antibodies from eBioscience (San Diego, CA), Biolegend (San Diego, CA) and BD: CD3e-APC-Cy7 (145-2C11), CD4-PerCP-Cy5.5 (RM4-5), CD8a-PE (53-6.7), ICOS-PE-Cy7 (C398.4A), CD69-BV421 (MIH5), CD19- PE-Cy5(6D5), CD138-PE-Cy7 (281-2), B220-APC-Cy7 (RA3632), CD80-PE (16-10A1), CD86-APC (GL1), CD3e-FITC (145-2C1), CD11b- PE-Cy7 (M1/70), Ly-6C-PerCP-Cy5.5 (HK1.4), F4/80- APC-Cy7 (3M8), CD192/CCR2-BV421 (SA203G11), CX3CR1-APC (SA011F11), CD135-BV421 (A2F10.1), CD11c- PerCP-Cy5.5 (HL3), Gr-1-FITC (RB6-8C5), Ly-6G- APC-Cy7 (1A8), CD287/TLR7-PE (A94B10), CD103-PE (M290), IFN-γ-APC (XMG1.2), IL-4-PE (11B11), IL-2-PE (JES6-5H4), IL-6-APC (MP5-20F3), IL-10-PE (JES5-16E3), IL-13-eFlour450 (ebio13A), IL-17-PE (TC11-18H10.1)), IL-21-APC (FFA21) and their corresponding isotype controls.

Flow cytometry of aortas and whole blood was performed as described previously [33 (link), 68 (link)]. Briefly, cleaned aortic vessels were digested with liberase™ (1 mg/mL, Roche, Basel, Switzerland) for 30 min at 37 °C and passed through a cell strainer (70 μm) to yield a single-cell suspension. Single-cell suspensions were treated with Fc-block (anti-CD16/CD32), washed, and surface stained with CD45 APC-efluor 780 (#47-0451-82, 2 µg/mL), NK1.1 PE-Cy7 (#25-5941-81, 2 µg/mL), F4/80 APC (#17-4801-82, 4 µg/mL) from eBioscience (San Diego, CA) and TCR-β V450 (#560706, 2 µg/mL), CD11b PE (#553311, 2 µg/mL), Ly6G FITC (#551460, 5 µg/mL), and Ly6C PerCP-Cy.5.5 (#560525, 2 µg/mL) from BD Biosciences (San Diego, CA). Dead cells were excluded by staining with Fixable Viability Dye eFluor506 (#65–0866-14, 1:1000, eBioscience, San Diego, CA). Based on a live gate, events were acquired and analyzed using a BD FACS CANTO II flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and FACSDiva software (Becton Dickinson, Franklin Lakes, NJ), respectively. Gating was performed according to a previously published strategy [63 (link)].

To quantify biofilm-associated leukocytes in the soft tissue surrounding the infected knee, homogenates were filtered (35 μm; BD Falcon, BD Biosciences) and RBCs eliminated using RBC Lysis Buffer (Cat #420301, BioLegend). Cells were washed and incubated with TruStain FcX (Cat #101320, BioLegend) and stained with Live/Dead Fixable Blue Dead Cell Stain (Cat #L23105, Invitrogen), CD45-PacBlue (Cat #103126; RRID:AB_493535), Ly6G-PE (Cat #127608; RRID:AB_1186099), F4/80-PE-Cy7 (Cat #123114; RRID:AB_893478), and CD11b-FITC (Cat #101206; RRID:AB_312789) (all from BioLegend), and Ly6C-PerCP-Cy5.5 (Cat #560525, BD Pharmingen). ROS production was assessed using a CellROX Green kit (Cat #C10492, ThermoFisher) according to the manufacturer’s instructions. For individual samples, 10,000–100,000 events were analyzed using BD FACSDiva software (RRID:SCR_001456) with cell populations expressed as the percentage of total viable CD45⁺ leukocytes as previously described [15 (link)].

Animals were anesthetized at week 2 after transplantation by intraperitoneal injection of phenobarbital (10 mg/kg), then 100 μl peripheral blood, bone marrow and 1 mg spleen tissues were harvested. Single cell suspensions were generated from bone marrow and spleen tissues by mechanical disruption using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) for 1 min. Blood, bone marrow and spleen cell suspensions were then suspended in 0.83% ammonium chloride solution containing 10% (v/v) Tris buffer (pH 7.65) to lyse erythrocytes. The cell suspension was centrifuged at 400 × g for 10 min at 4 °C and purified by density centrifugation. Cell preparations were stained using the following fluorochrome-conjugated antibodies: CD11b-FITC, CD115-PE, Ly6C-PERCP-Cy5.5 (BD Biosciences, San Jose, CA, USA).