Anti ly6c percp cy5

Characterization of immune cell subsets in the liver was performed essentially as described previously^{21 (link)}. The individual samples were analyzed with a LSRII/Fortessa flow cytometer (BD Biosciences) and the FlowJo software Vx (Treestar). All indicated antibodies and reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using cytometer setup and tracking beads (BD). Single cell suspensions were created using the Miltenyi Liver Dissociation Kit (No. 130-105-807) and the GentleMACS isolator (Miltenyi) using standard protocols. The following antibodies were used: anti-CD3-PE-CF594, anti-CD4-V500, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326 (EPCAM)-BV711, anti-Ly6C-PerCP-Cy5.5 (all from BD), anti-CD8-eFluor650, anti-CD11b-eFluor605NC (eBioscience), anti-CD45-VioBlue, anti-CD49b-PE, anti-MHC-II-APC (Miltenyi), anti-F4/80-PE-Cy7, anti-Ly6G-APC-Cy7 (Biolegend). A gating strategy is provided in the supplementary material and methods (Fig. S1).

Splenocytes isolated from control and TCL1 leukemia-bearing mice were suspended at a density of 4 × 10⁶ cells/ml, incubated with 1.0 µM CM-H₂-DCFDA fluorescent probe (Molecular Probes, Eugene, OR, USA) in PBS at 37 °C for 30 min and then washed with PBS. Next, for distinguishing the population of living granulocytes, the cells were incubated with a Zombie Aqua™ Fixable Viability Kit (BioLegend) for 20 min, washed with PBS and subsequently stained with the following fluorochrome-conjugated antibodies: anti-CD11b-PE (eBiosciences, San Diego, CA, USA), anti-Ly6C-PerCP-Cy5.5 and anti-Ly6G-APC-Cy7 (both from BD Bioscience). After a final wash with PBS, to determine the intracellular ROS levels, the geometric mean fluorescence intensity (MFI) of oxidized CM-H₂-DCFDA in CD11b⁺ Ly6G⁺ cells was analyzed by flow cytometry.

Characterization of immune cell subsets was performed essentially as described previously (20 (link)). Samples were acquired with a LSRII/Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software Vx (Treestar). All antibodies and secondary reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking beads (BD). For characterization of immune cell subsets, the following antibodies were used: anti-CD3-PE-CF594, anti-CD4-BV711, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326-BV711, anti-Ly-6C-PerCP-Cy5.5, anti-NK1.1-BV510 (all from BD Biosciences), anti-CD8-BV650, anti-CD11b-BV605, anti-F4/80-PE-Cy7, anti-GITR-FITC, anti-Ly-6G-APC-Cy7 (from BioLegend), anti-CD31-PE-Cy7, anti-CD117-APC-eFluor780 (from eBioscience), anti-CD45-VioBlue, and anti-HLA-DR-APC (from Miltenyi).